genomlc library construction and analysis - hem · 2. ute the matrix below as a checldist while...
TRANSCRIPT
Construction and Analysis
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LibraryGenomlc
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Construction of aGenomie Library of
Å DNA
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A genomic library il the collection of an organilm 'I total genetic comple-ment. ChromOlOmal DNA il typically extracted and digested with ODe ormote restrictiOD enzymes. The resuJting restrictiOD fragments are ligatedinto a vector-usuaUy a modified bacteriophase or yeaa chromosome-and tranaformed into an appropnate hOlt ceU line. The genomic librarymen CODIisu of tbouaands of plaques or host alla of tranaformantl, eachof which potentiaUy containa a different fragment of chromOlOlD81 DNA.The library can be repeatedJy screened by hybridizatioD to identify thosehast ceUs mat have taken up a vector containing a sene or DNA sequenceof intaat.
The term 1/ibrary usuaUy refen to a gmomic library in which eukary-otk DNA il dODed into a modified 1 vector. In tbillaboratory, however,the 1 genome il doned inta a plaamid vector. In Part A, ..Rtstriction Diseatof Å and Plaamid pBLU,. lampa of genomic and vectOr DNA are eachdouble-digested with &"rID and HindIIJ. FoUowing incubation at 37"'c,samples of the DNA are e1ectrophoresed in an aprose gel to confirmproper rotting. The double diFst of 1 DNA generates 13 &agments, rang-mg in Iize from 125 to 16,841 base pain. Plasmid pBLU haJ a lingIerecognitioD lite for each enzyme, located ooly a kw bue pain apart,within a doning lite caJJed a "polylinJcer.. The double digest opena theplasmid by removing tbc short sequence between tbc BamID and HindIIIätes.
In Part B, "Ligation of 1 and pBLU Ratriction Fragments, . the restric-
tion digests of Å and pBLU are fint heated to destroy restrictioD eozymeactivity. Samples from each reaction are mixed together and incubated at
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2 2 O LaboratorY '4
room temperature in a buffer containing DNA ligase, ATP, and magne-sium. Tbe BamHI and HindIII "sticky enda- hydrogen-bond with theircomplementary sequences ta align restriction fragments. Lipse catalyzesthe formation of covalent phosphodiester bonda mat link complementaryenda inta stable recombinant-DNA molecules.
DlgataI. Set Up R88trfctlon
ADO Dlg..ted Dlg..ed1~ p8lU .~BlU
. Bu".. BamHI/HtldIl
). DNAlUfeJ . .
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II. C88t 0.8% Agaf'088 Gel
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TRANSFER Dlg..ted S8mpl. Dlg..ted Sample88npIe8 . 1 1 pBlU pBLU
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. IV. Staln Gel with Ethldlum Bromide, VIew, .nd Photogr8ph
STAINgel
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L.IIbo. 81M, 14IP8rt ARestrtalon DIpst of Å and PtIsmkI p8lU
INCUBATEMIX
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CONTINUE INCUBA 11ONof DIg..ted a. 8MDlgeetedpBLU
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PHOTOORAPHget r
VIEW
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. ..,---The proc:ea of constructing and analyzing recombinant -DNA molecules inLaboratories t4-t7 is not trivial. However, good reaultl can be expected,if the directions are foUowed carefully. Almost any plasmid containing a8dectable antibiotic-resistance marker can be used to demonstrate thedoning of foreign DNA 8equences into E. coli. Thia experimental unit hasbeen optimized for the plasmid pBLU, and the extenaive anaJysis of resu1tsis bued entirdy on recombinant molecu1es derived &om this patent pw-mid. It is un1ike1y mat your in8tructor will substitute pBLU with otherplasmida, because pBLU was constructed specificaUy as a teaching pla.-mid. It offers the foUowing advantaga:
t. pBLU is derived &om a pUC expression vector mat replicates to ahigh number of copies per cell. Yields &om plasmid preparations aresipificandy pater tban those obtained with pBR322 and otherplasmids with a lowcr copy number.
2. The pUC plaamids encode a partiallacZ gene product mat must becomplemented by a corresponding partiallacZ product encoded bythe bO8t cell. pBLU contains the mtire lacZ geM and requires noboat cell complementation. 'Therefore, &:1Y kuZ minus a holt ( a bost
W 11ft ii - 1acIång a functionallacZ gene) can be used; recommended strains:/rt-tld: ~~a":'~ includeJMtOt and]MtO7.I o't(Jldt nr ..f14 3. pBLU does not have IacI repressor activity, wbich e1iminates the need
. t , " '"' for the inducer IPTG in the growtb medium.~ h/f ~ ./
,;/ Jr,;:; i. :The PnKIent ControI
, ,I;",.. //t..lt. hl~" ~ ,'In Section In, samples of the restriction digests are electropboraed, before
" liption, to confinn complete cutting by the endonucleases. Thia prudent
conttoi is standard experimental procedure. If you are pressed for time,,. your instructor mayadvise you to omit electropboresis and ligate DNA
directJy foUowing the restriction digest. However, be sure to pretest meactivity of the BamHl and HindDI enzymes, and incubate lang enougb for
L complete dipstion.
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PRELAB NOTES
-DNA Restriction and Elec-Review the prelab nota in LaboratOry 3,trophoraia. II
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ConstnICtion of a Genomic lJnry of ~ DNA 223u u u u uu u u..u u........................
~ ~~..~..~.......Por dirat: 0.5-10-t&! miaopipemx + tipsr ~ JIMd I DNA . 1 0-1 00-t&I miaopipettor + tips
~S' ~ J.IWI pBLU f'lC! 1.5-ml tubes&rmHII HiJIdDI aluminum foi1be rattiaioo bufler beakcr for wucrIuIed tips
camera and film (optioaaJ)For .J.arophorais: diaposabJe pava
0.10 ~ I DNA dcctropborcsiI box
0.1 O ~ pBW maskins tape10IIdiaa dye. miaofu. (opåoDal)1.0% apI'OIe ~ ,- PanJilm or waxed paper (optiooaI)1>< TriaIBoraRIIDTA (TBE) buffer permanent marker1 P&'mI ecbidium bromide plasåc: wrap (opcional)
power supplyFor Ueo~ test tube rKk
0.05 M KMnO. traDSilluminator0.25 N Ha 37"C water bam0.25 N N.OH 6Q°C water bam (!or aproee)
I. Set Up Restrictlon DI...( 1 O minutes;' tbcn 30+- minutes of incubation)
1. Obtain labeled reaction rubes containing Å and pBLU. Ute a perma-nent marker to aeld the label -digested" to each rube. Add other.reagmts dirKtIy to theu tIIbes.
2. Ute the matrix below as a checldist while adding reagents to eachreaction. U." fruh pipn lip for each reagmt.
IamHII1bbe Å DNA p8LU 2x Hlfldlll~.~.~ ~~~) = ~.~ """'" 3~
Digestcd pBLU - M pi (preadded) )L pi 3 piIt" Jd. 11. r ~
3. Alter adding all of the aoove reagents, dole the tops of the rubes.Pool and mix the rea8eDb by pulsing them in a miccofuge or bysharpIy tappina tbc bottom of the rube on the lab bench.
4. Place the reaction tuba in a 3rC water bath, and äncubate tbem for30 minutes or longer.
Por di,lat:. ~HIJ&1/DNA ., ~~pBLU yM(f
&ImHII HiItdIJI2x rattic:tioo buffer
e/t~fts
r .MaJ opbotwis:0.10 JIIfp1/ DNA0.10 JIIfp1 pBLU
Ioedia& dye1.0% 8plOIe { ,-1>< TrillBcJn8rlEDTA (fBE) buffer
1 PWml ethidiwn bromide
Porl
Por tkcOlltlllrfiNllio0.05 M KMnO.0.25 N Ha0.25 N N.OH
RestrictionI. Set Up
( 10 minutea;' then 30+ minutcs of incubation)
After a {IIJl30-minute ina4batiorJ (or lorrger), freeu the reacticms at-20.C Ulflil yOM are ~aåy to amtinueo Tbaw the reactioru before pro-ueding cm to Sectiorr III, step lo
O.5-10-tL1 miaopipettor + b..10-1001&l micropipettor + bp'
l.S-ml rubelaluminum foilbeaker for wuu:IuIed tip8camera and film (optionaJ)
disposabJe glova~boxmaakingtapemiaofuge (optioDaJ)ParafiIm or waxed paper (optional)permaucnt marker
plastic: wnp (opåonaI)power IUpplytest tUbe racktransiII umina ror37"C wuer bath60.C waler b.th (for aprose)
hkr to Laboratory 3, "DNA Ratric-rion and EJearuphomis," for detailedinscructioos on setting up reaajona.
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Rder to Laboratory 3, "DNA Resttic-tion and Elearophorais, .. for dctaiIedinstructions on c:utins and Ioading geL
II. cast 1.0% Agarose Gel
(U lDIDuta)1. CaRfully pour agarote solution inta the cuting tray ta 611 it ta a
depth of aboor S mm. The gel should cover only about ooe-dUrd ofthe hei8ht of the comb teetb.
2. Alter the aproee solidifies, place the gel-cutins tray inta the eIec-trophoresis box, and set up for eIcctropboraiJ.
UJWr rH .~ DOJC, IZIftII4W fIH1 pI "'"" '1011 an rway toamtiIrw. Th. pi will remain m good amditiorJ for sewraJ days if if is
complet.ly ~.J in buff.,.
III. Load Gel and Electrophorese
(10 minutes; tben 20+ minutes of eIectropboresia),.. t '.' ,.t.. .99...' t9YO t. ", t .'-'lUT II u~vu ". UR; """"U&M.unuua ~.. v. "" 8UU YUL.U IIU: II:~-
trophoraed to determine whether the DNA. are complecely cut. Thesereatricted samplCl are electrophoresed along with uncut Ä and pBLU asCODtrola.
1. u.e a permanent marker to label (Wo clean loS-mi rubes .88IDp1e Å"and .88IDp1e pBLU.. Remove the .digested Å. and .dipted pBLU.rubcs &om the 3rC w.ter heth. Transfer S J.&1 of DNA from the. digated Å" rube into the lO sample Å. tube. T ranafu S J.&1 of plaamid
from the "d~ pBLU. tube into the..u.mpJe pBLU. tube.2. Imm#!diately retJml the -di,ested Å. aM -dipst«J pBLU. tIIba to
the water bath. mul contmw incubatm, at 3rc "'""I eke-trophoresis.
3. Obtain l.S-ml tubea containing control Å and pBLU DNA.. Ute aperm-."..,t marker to adel the label -uncut8 to eacb tube.
4. Add 1 J.LI of loading dye to the digested -samples8 and undigested»contra"8 of Å and pBLU. Close the tape of the tubes and mix byeitber tapping the bottom of the tube on the lab bench, pipetting inand out, or puIsing in a microfuse.
S. Load the mm contentI of each sample tube into a separate weil inthe gel, as shown in the diagram below. Uu a frah pipet tip for eachsampk.
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Construction of a Genomic l.bwy or A DNA :1 15
6. EIectrophorae at 100-150 vala, anti! the bromopbeno1 blue bandahave moved )() 4() mm from the weUa.
7. Tum off the powcr supply, mnove the cutina my from the e1ec-tropboresil box, and mnakr the gel tO a dåapoaab1e wei8h boat (orother ahallow my) for --ini.
Iv. Stain Gel with Ethidium Bromide, VIeW, andPhotograph(10-15 minutes)
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Rftt8w the MCtIon on "&hIdIum 81 omkIe ScaJrq and ResponsIbIe H8ndHna"In ~ 8CIOo 'f 3. 'W8r dlspoable JIcw8 when ICIIInl.. vt8Wf... and pho-toanphq... and dun", deanup. Conftne all stIJNnI to . r.aia8d sink .....
1. Ftood the seJ with an ethidium bromide IOJution (1 Jllfml), andaUow åt to stain for 5-10 minuta. Dataia, if desired.
2. VJeW the gel LU1der1OUft:e.
Wlb _Idet I'" can dImaa8 )'CUl" ~ Nw8r look cINc:dy lit an unshWd8d LNlip soun:e whhout -re ... ot8Cdori. VI8w onIy ~ . fh8r or 88f8ty .......that lbIortJ the hannfuI ~
3. Make an expoI1Ue of 1-2 seconds with the camera aperature Id atfIS. Devdop the priot for the recom~ time (approximately 45eeamds at room temperature).
4. Take the time for proper clc:anup:
a. Wipe down the camera, the tranIilIuminator, and tbc Itainingarea.
b. Decontaminate the gel and any stainina solution tbat will not bereU8ed.
c. Wub your hands belme lenins the laboratory.
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or other uv Jishtan ultravio1ettranailluminator ~
'2 '26 I..aboratorv 14A
RESULTS AND DISCUSSION
Compare your stained gel with the ideal gel (below), and check to deter-mine wbetber the pBLU and ~ ONAs have been completely digested byBamHI and Hi,.dIII. Also refer to the restriction maps in Appendix 3 andpage so.
8ImI*p&U
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The sample pBLU laDe should showasmsk band of 5,<400 bp, usu-aUy in a position near the supercoiled form in the control pBLU lane.The presence of additional bands in the sample pBLU lane indicaressame degree of incomplete digestion. (Recal1 mat the digest actuaI1yproduces an additional 30-bp fragment, wbich quiclcJy runJ off ofthe end of the gel during electrophoresis.)
The sample Å lane should show 9-10 distiuct fragments, ranging in8ize from 493 to 16,841 bp; however, there is also a 125-bp frqmentdIat is olten too faint to be seen or else it runa off of the end of the gelduring electrophoresis. The digest actually produces a total of 13fragments, but several fragments are similar in size and do notresolve weil &om ODe another (eg. 2,409, 2,396, and 2,322 bp).Commercially available Å DNA is a mixture of bom linear and årcu-lar molecules, and this adds further confusion. Recal1 that the single-stranded COS sires at each end of the Å genome are complementaryand can liydrogen-bond to ODe another to form a closed circle. Apercentage of circu1ar molecules are covalently linked by phosphodi-ester bands. A cm at the leEt-most restricrion sire in the linear Å chro-
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Construction of a Genomic L..b-ary of 1 DNA 117................................................................................................................................................................................................................................................
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mosome (by BamHI) produces a S,50S-bp fragment; the right-mostsite (by HindIII) produces a 4,361-bp fragment. However,BamHIlHindDI digestion of the C;:OS circlet produces a 9,866-bpfragment, which is the sum of the left- and right-most restrictionfragments. Thus, the S,50S-bp and 4,361-bp bands mar appealfainter than expected, depending on the percentage of årcular mole-cu1es in the Å sample. (Hearing Å DNA for 10 minutes at 6S.C imme-diatdy befoR electrophoresis disruptl COS circles held oo1y byhydrogen bonds and increases the field of the end fragments; how-ever, hearing has no ~ on covaJently dosed cirdes.)
3. If both digesu look compJete or nearly compJete, continue on to Part
B, "Ligation of Å and pBLU Restricrion Fragments.. The reactionwill most li1ceIy have gone to completion with the additional incuba-tion during elec:trophoresis.
4. If either digest loob very incomplete, aeld another 1 J&1 ofBamHJ/HindIJI solution, and incubate for an additiona120 minutes.Then, continue on to Part B.
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Otg88ted Olgeated~ pBlU
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Dlg".~ pBLUUg But/ATP
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PRELAB NOTES
Review the prelab notel in Laboratory 78,Ratriction FragmenCl. .
For Further Information
The protocol presented here is baaed on the foUowing publishcd metbod:
Cobm, S. N.. A. C. Y. Cbaus. and H. Wo Boyer. 1973. ConItruaioo al bio&op:aJJyfunaionaI bacteriaI plasmida in vitro. Prouedm,. of tW Nmicmal Academyof sa..e. USA 70: 3240..I
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Ltrd~Step 1 is critica1: Heat denaturationinactivates the Jeatr8crion enzyma.
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For brief liptions of 2-4 houn. be sureto use a high-concentraåon IigaR withat Ieast S Weiss unitslf11 or 400 c:obe-aive-end unitsIJU.
LIGATION OF A. AND pBLURESTRICTION FRAGMENTS
R~. ... p s..ppIIeIl"~..~~.. """"'"
0.5-10-t11 micropipeuor + tips
1.5-m1 Nbcbeaker for wuceIuIed bp'
dåspo8able simarniaofuF (optionaI)tar Nbc rå70.C wacer batb
digesud I (&om Part A)ciigaud pBLU (&om Pan A)10>< liption buffa1ATPT4 DNA lipsedistiIIcd wata'
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Ugate A and pBLU(30 minutes; tben 2+ houn of lipåon)
l. 1nCUb8~ me -cupteO A. - ana -cupteO PDLU- NDeI ID a IU-~
water heth for 10 minutes.
2. Labe1 a clean 1.S-mI robe "ligation.. .... ,., ". ,
,j. Ute me mamx DeJOwas a CDecIWSt wnue aaa.ang reagentS tO mereaction. Uu a (resh pipet tip for e4Ch reagmt.
I Dt.-t8d I Ox Updon DNA'1\Ibe ..t)"~~"" Å . ,~~.. 8ufr8rtA,!",.. ..~.. ,,1JpM
Liption 4~ 4~ 2~ 8~ 2~
4. Aftr1: adding all of the above reagents, close the top of the tube. Pooland mix reagents by pulsing tbem in a miaofuge or by Ibarply tap-PinK the bottom of the tube on the lab beocb.
s. Incubate the reaction at room temperatule for 2-24 boun.- 6. If time permits, the ligatian can be confirmed by electropboresing S
~ of the ligatian reactian, along with BamHIlHindIII digests of Åand pBLU. Few, if any, of the low-molecuJu-weigbt bands in tbe Åand pBLU digem sbould be present in tbe ligated DNA gel Iane.Imtmd, the ligated Iane sbould show multiple bands of high-molecu-lar-weigbt DNA near the rop of the gel.
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Preeu the ligation at -20.C untiJ you are JWåy to corrtinue. Thaw thereactiom before proceeding on to Laboratory 1 S.
RESULTS AND DISCUSSIONLigation of the multiple Å fragments and pBLU produces a number of dif-ferent hybrid molecu1es, although not as manyasyou might expect. Eachclonable Å fragment must have ODe BamHl and ODe HmdID end, to com-bine with the BamHl and HindllI ends of the pBLU baåbooe and fonn acircular rccombinant plasmid. OoIy 5 of the 13 BamHVHmdIII &agments
Construction of a Genomic Linry of 1 DNA 23 I
from the linear l genomc satisfy thia requimuent: 493 bp, 714 bp, 2,396bp, ~409 bp, and 4,148 bp. A sixth clonab1e fragrnt'1t of 9,866 bp isderived from the eos circle. Multiple copia of a sinPe &agment or com-binatioDl of two different Å fragmentl can aJJO be lipted inta a siagIepBW backbooe. Three fragment plaunid, can be c:onatrUaed by combin-ing the pBLU badcbone with ODe of the five frqmenb baving ODe BamHIend and ODe HindID end and with ODe of the eight fragments baving twoBamHI ~ two HindIIJ enda. Consttucb with more tban ODe unit of pBLUan: aJao formed. 0nIy CODlttUCtl with at Ieut ODe unit of pBLU badcbooecontain an o. neceuary for replication and the ampicillin-resistancegene required for growtb on 8eIective media..
1. Make acaJe drawinp of all pocmtial two-tt.p-" (veaor + in8ert)recombinant plasmida.lndude the total bp Iize, fragment bp sizes,and the locations of B4mHl lita and HmdIII åta, the orisin, theampici11in-rai8t-~ aeae, and the lacZ aeoe.
2. Many different recombinant plasmicll are produced in dUa experi-ment. Draw severa! exampJes of recombiDant pl..mids that containthree fru:menll indudina an oriain of reolicatioa.
3. How many three-Eragment rec:ombinant pl8anw containins the7M-bp frqmeot can be COOItrUCIed?
-4. Deec:ribe how four !rapen.. can recombine to fonn a recombinantpWmid.,
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Transformation ofE. coli with A Library
In Part ~ -aa.ic Procedure for Preparing Compeceot CeUa,. E.. coli cellsare made competent to taJce up plalmid DNA by being incubated withcold calcium chloride. In Part ~ -Transformation of E. coli with Recombi-Dant M>BLU Plasmids, . competent cella are tranaformed with the liption
productl &om Laboratory 14. To prepare for replica platina in the lubee-quent laboratory, nylon membranes are plac:ed arop LB plares containingampicillin and the lactole analog X-gal. Samples of tranlfonned cells arespread on the plarea and incubated ovemight at 37.C. Tbe ampicillin inthe medium selectl for cells that have taken up plaamida and mat expressthe ampicillin-resiatance gene of pBLU. eeUa that take up a relipted pBLUplasmjd aJ.o expreu the liuZ gene producr, fi-galacrooåaae, whichhydrolyzes X-pl to form a blue product.
The BamHIlHindIII dige8t of pBLU in Laboratory 14 removes a 30-base-pair fragment from the lad gene but aves the origin of replicationand the ampicillin-resistance gene intact. Thua, aU pBLU recombinantsreplicate and express ampicillin resiltance. However, only rec:ombinantplasmida containång the 30-bp fragment regenerate an intact liuZ gene;transformants of thia molecule metabolize X-pl and give riJe to bluecolonies. Inserrion of a Å restriction ttalm1ent into the oBLU backbone di..n'pA thf' lfJt'.7. fl'nf'j trsano;:fnrm:IlnNl of hyhrid pR' ,,11. C'nn!lltn1l'ft dn nnt
merabolize X-ul and therefore!live me to whitf' {unmlorml cnlnniN,Tbe remaining laboratories in thi8 experimental unit analyze the Å
inseru found in plasfltidt &om these white colonies.
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T ransformBtion of E. coli with 1. lbrary 13 S................................................................................................................................................................................................................................................
PRELAB NOTES
Review the prelab nates in Laboratoriet 2A-C, S, and SAculture and transformation.
E coli StrainsIt is essenrial to use a WcZ minus E. coli strain tUt is unable to metabolizeX-gal. The protocols in this unit have been tested and optimized with thelad. minus strain }MtOt. Other WcZ minus ltI'8ins commonly used formolecu1ar biologica1 studies should give comparable results. However,gråwth properties of other E. coli stram. in suspension c:ulture mar differsignificandy--such as in the time needed to reach the mid-log pIwe and inthe cell number repracnted by optical densities.
Competent Cell Y1e1d
If competent cells are hems prepared in quantity for group WIe, remembermat 100 mi of mid-log culture yields 10 mi of competa1t cells, eno. for50 transformations (200 J&1 each).
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.For Further InfonnationThe protocol presented here is bascd on the following published metboda:
Hanaban, D. 1983. Studia on tranSformation of Esdurkhitl &o1i with plasmida.JOImIiIl ofM~ BioIogy 166: 557.
Hanahan, D. Techniqua for transformation of Bo coli. 1987. In D. Mo Glovcr(ed.), DNA Clorrhtg: A PraåU:al AfJproadI. ~ 1. Oxford: lIlL Praa.
Mandd, Mo and A. Hip. 1970. Calcium-dependeat bec:8aiopbaIse DNA in&cåon.JOIIf'1fIIl of MokaUar BioIogy 53: 159.
reprding E. coli
rolO Blank" tubes with 10 mi of water canbe U8ed for balance, if necdcd.S~ 1,000 )( g correspoods to2,~3,OOO rpm for most tabletopclinical centrifuges. A tight cell penetshould be visible at the bortom of thetube. If the pellet appears very lOOK oruncoosolidated, centrifugc for anotherS minutes.
~ OOrganize the materials on the labbench and plan out your sterile manip-ulations. If you are working as a team,ODe perIOD can handle the pipet, andthe other can rem~ve the cap and flamethe mouth of the robe.
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'{(f). A IV 9}f -fJ,~( l tf "'1" ~ b"r ) ~~ t~ ~ Iftt4r. ,r (Jt~, l r~, I .
,,
CLASSIC PROCEDURE FORPREPARING COMPETENT CELLS
. . .H~.~~.~~.H .H"""","'"~~ ~................s~ or 10-mlIteriJe pipeapipet &id or bulb100-1,0001&1 miaopipettor + ap.(or l-mi pipet)beaker of cruabed or cracked ice
beaker for wutW8ed up'
10%-bleach lIOIutioo or disinfectantbumer lIamedean papa' toWeIa
10 mi mid-IOIJMI01 cultureSO mM ca1åum ch1oride (Ca~)
clinica1 c:entrifuptest tube rack
Prepare Competent Cells
(~SO minutes)This enUr, IXfJlriment must be performsd utttur stmk conditions, usmgstIrik tuba and pipet tips.
1. Place a sterile tube of 50 mM Ca~ solution on ice.2. Obtain a U-mi tube with 10 mi of mid-Iog cells, and label it with
your name and the date.3. Securely dose the cap. and place the tube of cells in a baJanad con-
figuration with other classmates' tubes in the rotor of a clinica1 cm-trifuge. Centrifuge at 500-1,000 x g for 10 minutea to pellet the cellson the bottom of the culture tube.
4. Take care not to distu,b the cell pellet. Steri1e1y pour off the super-natant into a waste beaker for later disinfec:tion. Invert the culturerobe, and touch the mouth of the robe to a clean paper toWeI, to wickoff as much as possible of the remaining supematant. Flame themouth of the culture tube alter removing the top and apin beforerep1acina the top.
5. Sharply tap the bottom of the robe several times on the lab bench toloosen the cell pel1et.
~~. jft tO1/r. ,tJt<..
(Aht. 'I- !hi 8WiUV IfIIJ )
~u,.u~-.
1- ...
T ransbmItion of E. CDI with 1 Ubrwy 23 7"" """""""" ""-""" """"""-""""-"-'-"'-"--""-""_u"""", """"""
6. Vie a sterile 5- or 10-ml pipet to aeld S mi of ice-cold calcium chlo-ride to the culture robe. Briefly flame the pipet cylinder before uaingit. FIame the mouths of the ca1cium chIoride IUPPIy tube and the cuI-ture tube, aher removing the tops and apjn before replacina the
topL7. Clou tM cap of the atltur. tube tighu,. Immediately vortex the tube
to compleuly resuspend the peUeted ce1Is. I>uring vortexing, periodi-cally hold the robe up to the light to check on ita progress. Th. fin-ished aU suspmsicm shou/d be homogmeous. with no visible clMmpsof uu.. If a mechanical vortexer is unavailable, "finser vortex- ufo1IOWI:Hold the upper part of the robe eecurely betw~ your tbumb andindex finger. VJgorously hit the bottom of the robe with the index fin-ger of the opposite hand, to create . vorteX tbat lifts the cdl pelJct offof the bottom of the tube.
8. Return the robe to ia, and incubate for 20 minuta.
9. Following incubation, respin the cell. in a clinical centrifuge for Sminuas at S~ll000 x /l.
10. Talt~ special care not to disturb the diffuse ceU p~lIet. SteriJe1y pouroff the supematant inta a waste beaker for later disinfection. Invertthe culture tube, and touch the mouth of the tube to a dean papertowel, to wide off .. much al posaible of the remaining SUpem8t8Dt.Flame the mouth of the culture tube, alter removing the top andaa.in before reolaåna the too.- - - -
11. U. a tOO-t,OOO-,u micropipettor (or t-mI pipet) to steriJdy add1,000 J&l (t mI) of ~ ice-coId Ca~ to the tube. FIame the mouthof the cu1ture tube, after removing the top and again before replacing
---r'12. Clou the cap of the culture tuN tightJ,. Vortex to completely resuI-
pend the peUeted cells. The finished aU suspension should N homo-ge1UlOlll. with no visibk clMmps of cells.
Stort thtJ alls ;" a btJtIlt.n of ia ;" thtJ refrignator (at 1l/JP1'O%imat#yO'C) untiI you IWtJ ,tJady to lISiI thtJm. -s.so";,,g- at o-c (or as lem, III24 houri ;"cntIStJS thtJ comptJtMcy of alls (iw- to tm{olJ.
13. Take the time for proper deanup:
a. Segrep~ for proper disposaJ cuItwe platel and tubes, pipeu, udmicropipct tips that have come inta contact with E. coli.
b. Disinket the mid-Iog cu1rure, the tips, and the supematant fromstep'" and 10 with a 10%-bleach solution or a disinfectant (sucbal Lysol). .
c. Wipe down the lab beach with soapy water, a 10%-bleach IOlu-tion, or ~t.
d. Wash your handa before aving the laboratory.
~/It' IItf(lj#-~ ;'M)., 1141-wJu: ,eM-6\1w\t1 WA~ liM ~ ~ /f'~ r,t»~ "tt/;./" Ij.,~ "" :-t u. .f. ,.. 1,.- - .
~The cd) pellet will bec:ome inaasingIyd.i.fficuIt to R:lUlpcnd the lonser it sitsin the calåum cbloride. A complete cdIlU8pCDSion is probably the most impor-tant variable for obtaining competeDtcells.
öThe caJåum chIoride treatment .teerstbc adherilll propertia of tbc E. colicdl membranes. At dUa ooint. the c:eUpellet typicaJJy appears diffu.e or ring-8baped and .houJd rauspend mOl'eeuily.
o~The diffu8e ceU peUet shouJd rmupeudvery eu&Iy; do DO( overvortex it.
ö .
.!p(i~t)~ uff \~I--
~fJ,( r -.L ,. "'4 -rr)
/ J~rM /,Ittli J'itA.l
. tlr I)'V #,)r f)q' ~ ,
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)fl- Vi ,.
,~ Hd)
with AJpBLUE.collTransform
8
l8boratory I 5JPwt .T ransIormation of E cdI \ Recombinlnt ~wtm
Recomblnanta
{
PIum6ds
PRELAB NOTES
Review tbc prclab nOteS in LaboratOry S,of Bo coli with PIasmåd DNA. "
X-GaIThe chromogmic substrate X-pl (S-bromo-4-chloro-3-indoyl-D-plac:-
u. ,. I ,.tosade' 18 CJ1enucally reJatec.1 to A-pnospnate, one ot me SUDStratel ror eolordeveJopment in Laboratory 13, .Southem Hybridization of A DNA." Like~ntibiotia, X-pI il broken down by heat. When preparing agar platescontaining X-gaJ, make sure to cool the agar solution until the containercan be held comfortably in the hand (approximately 60.Q before addiogthe reagent. X-pl il expeoaive; ODe economica1 alternative to adding X-plto an entire batch of culture medium is to sterildy spread SO J.Ll of a 20-mglmlstock solution over the surface of an LB ampicillin plate. If time per-mib, anow the X-pl solution to diffuae into the medium ovemight orincubate it at 3rc for leVer'al hourI before UIC.
Nylon Membranes for Replica Pladng. I" ., . ,. ,. ... , r., ,,' .. .m pccparaaon ror LaDOrauxy IGA, ..~epUCI 1I1IDaR:I 01,., uonca,. aana-formed au, are spread anto a sterile nylon membrane, resring on the sur-face of an LB agar plate containing ampicillin and X-gal. Technica1Jy,nylon membranes used for replica plaring shouJd be sterile. However, thefresh dean membranes provided by the manufacrwer are rarely contami-nated and can be used without sterilization. This is another application ofMax Delbriick'. principle of -limited sloppinesa- in molecular biology: ItusuaUy d~ not harm resulta, and anribiotic se1ection helps compensatefor åt. If you wish to be scrupuloua, sandwich tbe membranes individuallybetween sheetl of filter paper, and autodave tbem.
In typical protocola, a dry membrane is placed directly on the surface ofa fresh agar plate. However, this simple method can lead to uneven wettinsand crumbling of the membrane, especially when relatively dry plarea areheing used. We recommend thoroughly prewetting eacb nylon membraneby submerging it in a culture plate containing 10 mi of sterile LB media ina petri dåsh. Since nyloo membranes are somewbat expenIive, they are norplaced on conb'ol platel that will not undergo replica traD8fet.(8For Further Infonnadon
The orotocol oresented here is bascd on the followina oublisbcd mctboda:
Cohea., S. N.. A. C. Y. aw.. L lUa. 1912. NonchrolllOlOmal anribiobc rai8-tance ån baeten.: Genebc: transformation of EscherichitJ coli by r-fKtorDNA. ProcnJings of th, Natiottal ~ of ScMru:es 69: 2110.
Grunstein, M. and D. S. Hogne8L 1975. Colony bybridization: A metbod for theiIolation of cloned DNA. that contaån a apecific gene. ProueJirt" of th,Natiottal AauImry of sa.u. USA 12: 3961.
Lederberz, J. and E. M. I.ederbeq. 1952. Replica plating and indirect eeIectioa ofbacteria1 mutaDes. JOIIt'NIl of &amoIogy 63: 399.
Messing. J. and J. VIeira. 1982. The pUC plasmida, an M13mp7-derived syltanfor insertiOD mutagenesia and sequencing with syntbetic uni\'enal primen.GerM 19: 25'.
Colony Transformation"Rapid
2 4 O l.aborItOI y 15 B
u.,.u;I J .-
-~ tr(.!i~/oJ} 7
~ Im. l2JD'1/ItILJJ
Jb.t~ iJ-. J~ ~ JWf Ll;""~.
~~ ~Iil i ~~il ~M frfl(
~UU! a frah lip to pipet each ~ ofDNA. Opcimally, ftame the moUth ofthe c:u1turc tubes aftu mnoving theropa and again before replacing therops.
Store the ttmaindet of the Jjgated DNAat ".C in preparation for eIec-tropbomic analysis in Laboratory 17.
Cultur8, Medla, and RupntI $uppi. lind Equlpment, .. ... ' """""""""
competmt I:.. COlI CC1IIID'Om rart Al !~!W1U nuaopIpamI"" apI
lipted .vpBLU (from Laboratory 1<48) 100-1()()()1&l micropipettor+tipI0.005 f1MI1 pBLU 0.5-1 ().p1 miaopipeaor + tipILB brotb 2 15 -mi culture tube8four LBlamp + X-pI pIata beabr of auabed or cncbd icetwo nylon membraDa beaJc.er for wutcIu8ed tipILB brom in ~ place -biobq- or hcayY-duty ttUh bq
10%-bIe8cb lOIutioo or disin&aant(such u LyIoI)bumer Samecell apreader
diapouble PmaforcepspertIWIeDt marker~ tube rack3rC iacubator3rC sbaJång water batb (optionaI).U.C watu bath
(
Transfonn E coli with AJpBLU Recombinants
(70-90 minutes)
This entire experiment must be performed under sterile conditions, usingmrik tuba and pip« tips.
1. UIe a permanent marker to 1abe1 two sterile tS-mI culture tubes:
+pUG -ligated WpBLU+pBLU - pBLU control
(,
(,
2. Add 200 JIl of compctent celJa to each tube. Return the J'f'nuliningcompetent ceUs to ic:e, and store tbem at O.c.
3. Place both tuba on ice.
4. Add 10 JIl of ligated ÄlpBLU direaly mm the uU suspmsion in the+pUG tube.
S. Adel 10 JIl of 0.005 J18/JIl pBLU direaly mm the uU suspmsion inthe +pBLU tube.
6. Lighdy tap each tube with your finger to mix the solution. Avoidmaking bubbles in the suspension or splashing the suspension up onthe side of the tube.
7 ~ Hum hnf.h t'llh- tn i~ fnr ,n ",in.--8. White the celJJ are incubating on ice, WIe a permanent marker to
label four LBlamp + X-gal plates with your name and the date.Divide the plates into pain, and mark tbem u foUows:
+pLig: Mark ODe platC UO and the other plate Ltoo.+pBLU: Mark ODe platC 820 and the other plate B100.
,t
Using gloved handt or forceps, prewet a nylon membrane by sub-merging it in sterile LB broth containcd within a cu1turc plate. Wbcnthe mcmbrane has been completeJy wcncd, carefully place åt on theagar surface of the UO plate. Avoid trapping air bubbla behHathe membrane and the agar sur(au.
9.
WET membr- wfCh L8 In
Id 01 pI8rI dI8h
PIPET cell SUlp8lIIIononID ~.
eolanJ Men""".. WIItdI.
10. Wet a second membranet as described ahave, and place it on tbc agarsurface.of the LtOO plate.
11. Fol1owing the 20-minute incubation on icet heat &hack tbc celIa inboth rubeL The cells must receiw Il shaTp and dist#u:t sbod..a. Carry tbc: ice beaker to a 42.C water heth. Remove the tuba &om
the ic:e, and immediately immerse them in the watu bath for 90~.--------
b. Immediarely return botb tubes to ic:e, and Jet them stand fot' at.. '.--- & &uua... &&&AUU_.
12. Add 800 ,u of LB broth to eacb tube.1.ighdy rap the robes with yourfinger to mix the solution.
13. Allow the ceUJ to recover by incubating botb tubes at 3'-C in 8
shaJdng watu bath (with moderate agitation) for 20-40 minUteI.
The alls am w alZow.d to recowr fm' Ullef'aI hows. A longer reaweryperiod can help to compmsate for tI poor ligatioft or for cells of lowcompetenu .
Af,.
, l.. I./!,~lo .1I .. I
LA Y W8tI8d membrIIl8 onl.B/8mp ~ pIIte
SPREAD cell lUIp8I'1Iion
ö~
The recoYa')' inaeases the number oftransformants dIat express me mil-taDCC protein by two 10 fourfoId, bot ircan be omined if tbcre are time COD-Stramts.
~..-..
242 l.abOI atOf v I S8
The added 1.8 broth increallCl the totalvolwne of Ijquid 00 each platc to hdpto evenly spread the small volume (20~) of transformed cdls on the UO and820 platcs.
Do not aDow the cells to sit too 10118 onthe plata before spreading mem. Theobject is to evenly disuibute and sepa-rate the transformed cells so mat eachgives risc to a disrinct coJony of cloRa.
The spreader, submcrgcd in alc:ohol, i.alread)' sterile. TItus. the onJy purposen& ~"- II i. ~n hnm nff rh.. ..lrnhnl-- n --- --- _n__-_-
before spreading. "The spreader wiUbecome too hot if it is held direc:tly inthe burner flame for severa! seconds,and this mar kill E. coli cells on theplate.
i
TaJte c:aft not to gougc tbc agar or themembranc.
~
14. UIe the matrix belowas checklist as the +pLig and +pBLU cellJ arespread on plates in the foUowing steps.
I . It: MdU I I. - - r-- r-- .""""""""""""""""""""""""""""""""""""""""""""""""'-"""""-"""'" .-....................",n on..! "n..! --- --,...LtOO - 100 J&1 -~ ~J&1 - WJ&1Bl00 - - 100 J&1
IS. Add 80 J.LI of sterile LB broth to the centen of the UO and 820plates. Then use fresh tips:
a. Add 20 J.LI of cells from the +pLis tubc to tbc LB broth on the UOplate.
b. Add 20 J.LI of cells from the +pBLU tubc to the LB broth on the820 nIa~.-
16. Use II fresh lip to aeld 100 ~ of cells from the +pLig tube to the Ll00...1..-'
17. U. II frah tip to add 100 J1l of cells &om the +pBLU robe to the"'.o . .U.&VV 1'18""'.
18. Sterilize a cell spreada; and spread the celJ. over the surface of each,.. .. .
or the tour platl:l m SuccesllOn.
a. Dip the spreader into the ethanol beaker and briefty pass itthrougb a bumer fJame to ignite the alcobol. Allow the alcobol tobum off tIWtIY from the bumer fIanw.
Be extnmely careful not to ianlte the ethanoIln thebeIker.
b. Lift the plate lid, like a elam shell, only enough to allow spread-äng. !
c. Cool the spreader by touching it to the agar, to the membrane lur-face away &om the cells, or to the condensed wafer on the platelid.
d. loucn me spreaaer to me cell suspenston, and genöy drag If back rand Eorth several timea ac:roa the agar or membrane surface.Rotate the plate one-quartu tum, and repeat the spreadingmotion.
e. Replace the plate lid. Return the cell spreader to the edw1o1 with-outflamina.
1 Q R ~ ..h.. ..ft_~- nfttL".. n... h..1n... i.. ~........-- - n --- _, n __n- _n__- _n __n_-
20. Ler the plates set for severaJ minutes to allow the ce11luapensåoDS to. . ..-- .----..--.21. ~rap. the..platet t~~~ wi~ ta~, p~ ~ upside down in a
.j r~ U1CUDatOr, ana ~ mem ror .u-4"t~ours. .
22. Alter the initial incubation, store the plates at 4J.C to arrat E. coligrowtb and to slow the growth of any contaminating microbes. St.rweither the 1..20 or the L1 00 plate to bep uboratory 16.
..
,I
Tab the time ~ proper deanup:a. Segregate for proper disposal cu1ture plates and tubes, pipen, and
micropipettor tips that have come inta CODtact with Bo colL
23.
b. Disinfect tbe cell suspensions, the tubel, and the tips ovemightwith 8 10%-bleach solution or disinfectant (such u lyd),
c. Wipe down the lab bench with soapy water, a 10%-bleach sol u-boa, or disinfa:tant.
eL Wash your hands before leavins the laboratory. .1...
~ESULTS AND DISCUS'SIONAunt the numhet of blue and white colonies growjng on each plate. UIe a~ent marker to mark the bottom of the culture plate as eacb colonyI counted. A total of 50-200 colonies sbould be obeerved on the UOxperimental plare, with about equal nwnbers of blue and white ooloniea.~pproximately live times as many colonies should be observed on the 820ontrol plate-atl, or nearly aD, blue. The corresponding LtOO and 8100,lates should contain approximately live t:imes as many colonies. Anxtended recovery period (step 13) would most likely inflate tbesexpecu:d rauID.
Blue colonies may be pale in coloc and difficult to diltinpish &om,hite colonies. Refrigerating the plates for an hour or more can help toltensify the color. If the plates are overincubated at 3rC or left at room=mperature for several days, small, white -..tellite- colonies mar bebserved growing in s circle around large, resiaunt oolonies. Satelliteolonies are donet of the ampicillin-sensitive cells tbat begin to grow asle antibiotic is gradually reduced below the selection tbreshold.
1. Record your colony oounts for each plste in the matrix below. Werethe results as you expected? Explain the poaible reasons for varia-tions from the expeaed results.
White CoIonI8I Blue CoIonI88.." "'" """"""""'"
tO
LOO
~O
100
2. Campare and contrast the growtb on esch of the fonowing pain ofplates. What does esch pair of plates ten you about transformation,antibiotic selection, and doning efficiency?
I. Total colonies on the UO versus the 820 (and dM Ll00 versus theBl00) plat.e
b. The percentage of blue (or white) colonies on the UO versus theB20 (and the LlOO versus the Bl 00) plat.e.
3. Explain why white satellite colonies appeal around a b,. colooy.
UO
LtOO
820
8100
(~
244
8
laboratory 158
4. Calculate the transformation effaciency of the controi pBLU transfor-mation (B20 or BlOO plate): the number of antibiotic-resistantcolonies per miaogram of plasmid DNA. The object is to determinethe mass of plasmid DNA that was spread on a plate and rhat wastherefore responsible for the number of transformants observed.
- .~ ... ~a. ueterDUDe me roull mass (m lIllI °T pDI...U U8eO m me traDGOrtI18-
tion - concentration of pBLU x volume of pBLU..- ., ,... ,,---b. 1Jeterm1ne the fTachon o, the Ull suspmsum spreaa onto the BZU
or B1oo plate = the volume of cell suspension spreadlthe total vol-
ume of the c:eU suspension.
c. Determine the mass of pBLU;" fbI! uU suspttns;on sprt!4d ontothe 820 or B100 plate = the total mua of pBLU (a) x the &actionof the c:eU suspension spread (b).
d. Expreu transformation e(fiamcy in 8cientific notation u thenumber of colonies per J.1I of pBLU - the number of colonies onthe BlO or BlOO platelthe mass of pBLU in the cell suspensionspread (c).
S. Ca1culate the transformation efficienc:y of the experimental tranafor-mation with ligated DNA (UO or LtOO plate).
a. Determine the total mall (m pg) of pBLU \lied in the initialrestriction digest (Laboratory t.A).. the coacentratioo of pBLU xthe volume of pBLU.
b. Determine the fraaion of pBLU digest used in the ligation reac-tion (Laboratory 14B) = the volume of pBLU digest in the liga-
tion/the total volume of pBLU digest.
c. Determine the (ractio.. of liBation reaction used for transforma-tion - the volume of the ligation reaction in thetransformation/the total volwne of the ligation reaction.
d. Determine the (Tamo,. of cell suspension spread onta the UO orLlOO plate - the volume of cellsuspenaion spreadlthe total vol-
ume of the cell suspension.
e. Determine the mass of pBLU in the cell suspension spruJ ontothe UO or L100 plate = the total maJS of pBLU (a) x &action b x&action c x &action d.
f. Express the transformation efficiency in scientific notation as thenumber of colooies per ~ of pBLU - the total number of coIoniesOD the UO or LtOO platelthe masa of pBLU in the cell suspension
~ 1_\. . .6. The "clasaic" transformation protocol osed in this laboratory typi-
cally achieves efficiencies of S x ur to 1 x 10- colonies per nUcro-gram. How do the efficiencies tbat you calculated in questioDl 4 andc ..."" "" .1.:. ".) ro ..,.,... --,.,.u...~. .1.. A:U )- --,.-- .- - O-' -- ,-- ~- --- -- ------.
7. How docs the length of postincubation recovery influence transfor-mation efficiency?
8. Why is high-efficiency uansformation important for constructing agenomic library of eukaryotic DNA?
For Further Research
1. Research d.iHuent raearch applicatioDl of the ~cto.idase 001-orimetric l)'IImI.
2. Design a series of experiments to detamine the optimum ratio ofreltricted Å to pBLU DNA for the liption in Laborator)' 1... Theobjective is to maximize the nomber of white colonia growins 00 anLBlamp X-gaI plate. For each experiment, rec:ord the total number ofcolonies on each plate and the ratio of white to blue coloniea. (Donot use nylon membrancs; a bybridization protoc:ol is IInNICnMry.)
3. Design an experiment to compare the transformation effiåenc:iea oflinear venus cin:u1ar pBLUA conscruaa.-
4. Design an experiment to detmnine the fraction of cella that took upplumid DNA.
i
,
~
T ransbmatIon of E. coli with 1 UbrIry'-"""""'-""'-""""--"""""""0...0..0.00...00..o......... 245
of the ~cto.jdue 001-applicatioDl
(
\
'I.~
Colony Hybridizationof the A Libraryl
In dUa laboratory, a bybridization prorocol is used to screen the genomic Alibrary to identify dones containins the 784-bp BamIDIHindDI A &ag-ment. The starting point of the experiment is a nylon membrane contain-inB colonies &om either the UO or tbe L1 00 plate from Laboratory 15B.
Because the hybridization procedure requires lysing the E. coli coIonieaon the membrane, it il essential to work with a replica membrane derivedfrom tbe original. In Part ~ "Replica Transfer of A Oones," the mem-branc from either the UO or the L1 00 plate &om Laboratory 158 is sand-wiched with a &esb membrane. When the membranes are pressed togetber,sorne of each colony is transferred to the fresh membrane, creating a mir-ror image of the transformants on the replica. Tbe replica membrane isincubated at 3'rC for several hoors to alIow the E. coli colonies to grow toa suitable size for hybridization. The original membrane is ston:d at 4.C aaa source of dones for subeequent anaJysia.
Part B, "Hybridization of the Replica Membrane," begins a series ofsteps mat are analogoUI to those in Laboratory 13, .Soutbem Hybridiza-tion of A DNA." Firat, the membrane is exposed to a sodium hydroxidesolution that lyses E. coli cells on the membrane and denatures the cbro-mosomal and plasmid DNA. Following neutralization, the membrane isbaked at 70.C to fix the DNA to the membrane. The membrane is prehy-bridized with a blocking agent that binds to all areas of the membrane nota1ready occupied by bound DN~ t~us preventing the probe from bindingqonspecifically to tbe membrane. Then, the membrane is incubated withthe digoxigenin-Iabeled probe, whicb bybridiza to only those coloniescontaining ju complementary sequen~ a 2S-baae oligonucleotide withinthe 784-bp BamHVHindIII A &agment.
In Part C, .Nonradioactive Probe Detection," the hybridized ment-brane is washed to remove excess probe and incubated with the antidigox-igenin antibody/enzyme conjugate. The antibody binds to areas of the
.
.
/'
LAB~f60RY
248 Labo.atav 16
COLORIMEfflIC ~r8Y88llloc8IIon 01 ooIonI88 to whIchprab8 hybfIdIZ8c:t
~ . GenomIc lInry by CoIony Hybridlzadon
membrane where the digoxigenin-labeled probe ha. bonad to 8 recombi-Dant plasmid containing its complementary DNA sequence. AIka1ine phOl-
phatase is conjugated to the antibody. When this enzyme is incubated witha colorimetric substrate solution, it initiates a series of reactions that pro-duc:e a purplish-brown precipitate, reveating the clone5 containing theDNA sequena: of interest.
GROW coIonIet ofrecombin8nt pIunidI
~~0VERlA Y pIete withnylon membl'lllMl
§LIFT rnembr8ne ccnt8InlngnIpIIca of coIonie8W.. .-
Prep81'8 the Repla Uemb.... '", '.' '. ", - "",',,; ""..>':" ,,' ,~?,~~)~...~:
wno-.."'~- ,' i:,.'" ';". PLJa<rtQlr8.'m81~ ~"" \ ';."',: ',' . "..:1<:4("'1<:11 n.... ..' """'..'.., ""
..\I'!'~;~ ..', '.." , ~""., '~:~." }
" "", . .,~ ,.. ..,,.t... ...,,: '-"' ,' ,"',... " ,, "' .. ',~ .' ,"' ,. ". , .,r .,:. "' ,'" " . ",'. . ",. ~ O'
, '
. '." . '. ' , ." , ' , " ".. ~' ."' .Y' .-,~. ' . ;.t,..', ~t;-t~;~r,::; ~i' >;.~>~'<',~6\:" ';'\:'~ <.. ; ~ '. .,: < ~!~i;';;:tf.~ ~;~:~~.~'".,:,:"-:"-;o"'~.~'~""",,,,', ""'" ., '.. o
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MembranePrepar8
Labot 8tIory 16IPart ARepka T al Å CIoMI
f
250 l.aboratory I 6A
PRELAB NOTES
.Southem Transfer of ÅReview the prelab notes in Laboratory 138,Ilestriction Fragments. .
Wetting the Nylon Membrane
The replica membrane must be wetted before being placul in contact withthe original membrane. However, an overly wet membrane can causecolonies to run together, aH'ectin8 the precision of the an.aiy1ås. We recom-mend wetting the replica membrane by placing it on top of . moist agarpJate. If the agar p1a~ are old and re1atively dry, steri1e1y spread 100 J.L1 ofLB broth on the surface beforc wetting the membrane.
Filter Paper
All mention of filter paper in Part A and the enauing parti refen to Wbat-man 3mm filter paper, which is most commonly osed for oolony hybridiza-tian.
Felt
Fek (or a feltlike material) is used to help ensure uniform contact betweenthe membranes. Filter paper can be substituted.
For Further Information
The protocol presented here is based on the foUowing publisbed methods:
Grunmin, M. and D. S. Hognaa. 1'75. Colony bybridization: A metbod foc theisolarlon of doned DNAs mat contain a specific gene. ProCMdin,. of""NatioMl Acad.my of Scinas USA 72: 3961.
Lederbcrg, J. and E. M. Lederbers. 1'52. Replica platins and indirect sdectioo albacteria1 mutants.JOImfIIl ofBaamology 63: 399.
"'...- m.""""-- Equ---.. I lupp'.,.. ..or:'.'.~.~.~.." ".., .......................
UO or LtOO plate (from Laboratory ISB) ballpoinr paaLBlamp plalle cfisposable sima
feIt (12)( 14 cm)filter paper (12)( 14 cm)
flatforapl
LBlampplace
Prepare the Rep'lca Membrane
(15 minutes; tben several hOOR of incubation)
1. Using flat forceps or disposable gloves, obtain a freah nylon mem-brane: 11UJ is the replica membrane. Label the edge of the membranewith your initiala using a baUpoint pen. Wet the membrane by care-fuUy placing it OD the surface of the LBlamp plate. Cover the plate,and leave the replica membrane in contact with the agar until youare ready to WIe it in *P 4.
2. Use the forcepl to carefuUy pceI the membrane off of the UO orLlOO plate from Laboratory 15B (whichever ODe has 3~300 whitecolonies on åt): 1bia il the L mcmbraoe.
3. Place the L membrane, oolony side up, on top of six sheeta of fiItupa per.
4. Use the forceps to remove the repljca membrane &om the LBlampplate (step 1). CarefuUy align the edges of the wetted replica man-braue with the edges of the L membraue. Then, carefuUy lower thereplica membrane to contad the L membrane, so mat ther are super-impoaed as clOlely as pouible.
S. Place a square of fett (or filter paper) on top of the superimpolCCimcmbranea.
6. Place a gel-casting ttay (or book) o~ top of the filter paperlmem-brane/felt sandwich. Press firmly an,," evenly on the gel-casting tray(or book) foto aeveraI secooda.
7. Remove the gel-casting tray (or book) and the lelt. Use a sterile t8-gaose needJe to poke three holes througb the edge of the sandwichcdmembranes and into tbe filter papers below. The object is to make anasymmetric pattern of marks that will aDow tbe two membranes tobe reoriented alter tbey are separaced.
gd-<Utia8 hy ex booknyloo membraae (82 mm in diameb:r)sterile 18-p \IF needIe (or sc:i8on)3 re iDcu b8Ior
~Ncver touch the nylon membrane withyoor bare bands. Oils from your slånmay be depoaitaf on the membraneand can pnMDt tbc DNA from bindingfollowing ceU Iysis. Do Dot: fabel themembnme with a fdt-tip pen, bec:a URthe ink will bIeed wben jr ia 1m.
~It is important to align the replica andL membrana al dosdy u possiblc. Becareful, tboush; ooce the membnmacome inte coDfaCt, they cannot be ~sirioocd.
~Unevcn pralure can cause the mem-branes to slide against ODe another,smearing the colooia.
AJtemardy, Ute the lCiuors to alt 1eV-era) trianplar notches inta the edges ofthe membranes.
251
[i]~
Incubating the replica membrane on anLBlamp plate without X-gaJ will caUlepreviously blue colonies to appearwbi~ This is desirabIe. because bluecolonies can be nUstaken fCK purplehybridizaåon .......Incubate onJy lons enough to obtain E.coli col0nie8 of 1-2 mm in diameter.Do not cm:rincubaIe: Satellite coloniesmar appeal to compLicate the compari-100 with tbc original membraoe.
8. Using gloved hands and forceps, cacefully peel the two membranetapart. Return the L membrane to itl original plate (with the coloniesfacing up) and store it at .. .C.
9. Return tbe replica membrane to the LBlamp platt from step 1 (withtbe colonies faång up), invert the platt, and incubatt at Jr<: for 1eV-eral houn or overniabt.
RepIIca 1TI8IT1br8.
PREWET ~
l1'I8fTIbr-. ~dl8h
! PRESS
Lmembr8. Jr
ö~
PlACII,epllCa .
rnerntJrwwonll'lf D8n8turtng
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fUC&repllcame..Jb.-on nr Neulr8llz8llOn
~;.. i;,.
.j: .; " BAKE
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PAEHYBAIOIZE
~
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HYBRIDIZI
8...
~
Laboratory 16IP8rt .HybridIwIon al die RepIca MembnI.
DAYon"~.~AJRDRY
(1;
~
-
".
254 LaboratoryI68
PRELAB NOTES
Review the prelab nates in Laboratory 13c, -HybridizatiOD of the Ä.Probe. .
Batch Processing
All replica membranes are processed together in a -batch" procedure,requiring preparation of just ODe appropriate1y sized contaioer of eachreagent.
For Further Infonnadon
The protocol presented here is based on the foUowing pubtished methoda:
Grunstein, M. and D. S. Hognaa. 1975. Colooy hybridizatioa: A method for theisolation of cloned DNA. mat contain a apccific gene. Procudings of th.Natiorral ACIIIlmwy of sa...c. USA 72: 3961.
Latbe, J. 1985. Syntbcdc oligonucleotidc probe8 deduced &om amiDO aådaequcnce data: ThcoIetical and practia1 c:onsåderatioaa. ]0tInIiIJ of MoIeat-"" B;ology 183: 1.
Manin, R., C. Hoover, S. Grimme, C. Grogan, J. HoItkc, and C. KeuIer. 1990. AbigbJy sensitive. nonradioactive DNA labeq and dm=c:tion .)'Item.BioT~ 9(6): 762.
CoIony Hybridization of 1 LJnry 2 5 5
HYBRIDIZATION OF THE REPLICAMEMBRANE
~~~ ~.CI~~.~.~~ .....baUpoint pendisposable gIovafilter paper (12 x 14 an)
flat forcepl
mulring tapeaven set at ro.cplastic cootaåner (15 x 15 x 4.5 an)pluåc cootaiDer (17 x 28 x 6.5 an)
SO.C shaking watcr batbtissue paper
tt'aY'
deuaturing buffaneutralizing bufferprehybridization buHerhybridization buHer
f
~
,.
i\
t
,,
r
...:
8. DenaturelNeutralize the Replica
(SO minutes; men 30+ minutes of baking)
1. Using forceps and gloved hands, place the replica membrane (colonyside up) on a filter paper saturated with denaturing buffel. Let åtstand for 15 minuca.
2. Transfer the membrane (colony side up) to der filter paper, and allowthe excess solution to drain off it for S minuteI.
3. Place the membrane (colony side up) on a filter paper saturated withneutralizing buffer. Let it stand for 15 minute8.
4. Transfer the membrane to a fresh pm of filter paper to dram excesssolution; then air dry it for 10 minuteI.
S. Sandwich the membrane between two fresh pieces of filter paper (12x 14 cm). Tape the four sides, and label the filter paper with yourname, using a soft lead penci.I or ballpoint pen.
I;J<.
J,
I
6. Bake the membrane at 70.C for at least 30 minutes.-After baking, the membrtm4 can be stored indefinitely at room
temperature.
~
Membrane
Never touc:h the nylon membrane withyour bare bands. OiJs from your skinmay be deposited on the membraneand can prevmt the DNA probe frombinding.
000000000000
~
2 5 6 l.aboratory I 68,,""""""""""""""""""""""""'"
~
2. BlOT bfIeIIy on dry 3 mm peper
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.. . Pt8ce membr8l1e between two 8heet8cA 3 mm p8p81", TAPE lides. and BAKE
3. NElITRAUZE on communIIITRtSt-I8CI ....
II. Prehybridize the Replica Membrane
(5 minutes; then 6()+ minutes of incubation)
1. Using gJoved hands, remove the nylon membrane from the filterpaper sandwich from Section I, and transfer it to a common con-tainer with prchybridization buffer. If the membrane sticks to the fil-ter paper, do not try to separate them. Place both in the prebybridU:a-tion buffer, and they will separate whm wet.
2. Incubate, with gende shaking, at SO.C for 60 minutes to ovemigbt.Cover the container to prevent evaporation.
(
III. Hybridize the Replica Membrane '" f~ PlVb~
(5 minutes; then 120+ minutes of incubation)
1. Using gloved hands, transfer the nylon membrane to the containerwith hybridization buffer.
2. Incubate, with gentle shaking, at SO.C for tWo houn to ovemigbt.Cover the container to prevent evaporation.
Waah and Develop Color
WASH
WIIhbtJI8r
WASK
POU"off
INCUBATE
AntJbodyI
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POUR .(I) on I
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Laboratory I "'-'t CNonradloaccM Probe Det8cdon
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WASH
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258 L.aboratory 16C""""",.0.0.00.0""0,..0.""""""""""""""""""""" "'0""""""",
8
8
PRELAB NOTES
Review the prelab nates in Laboratory 130, -Nonradioactive ProbeOetection,- and the instructions pertaining to eolor development mataccompany the Geniua 3 Nucleie Acid Oetection Kit. The numberedbuffers in this laboratory correspond to those in the Genius låt.
For Further InfonnatlonThe protocol presented here is based on the following published methoda:
Genius System UsUs Guide, Boehringer Mannheim Corporatioo, BiocbemicaIProduc:a, 9115 Hague Road, P.O. Box 50'U4, IndianapoUa. IN 46250-0414.
Manin, R.., C. Hoover, S. Grimme, C. Grogao, J. Holtke, and C. KeaIer. 1990. Ab.ighIy IeDlitivc, nonradioutiw DNA labdia& and dctectioo ly'Item.
BioTedmiqNa 9(6): 762.
,
)
j.
,
.
).f
~
Colony Hybridization of~ Ubrary 259'MM M""M "'M """"""""""""""""""""""""""""""""""""""""""""""-..........................................
RADIOACTlVE PRO BEECTION
EquIpment 8nd lupp'.. ... ..................................
buffel (2>< SSc, 0.1 % SDS) disposable gIovabufkr...uO.1M Tris pH 7.5, O.15M filter p8per~ flat forcepe~~ulfer 1 + 1'" wlv blockius petri diab (150 mm)nqcar anaI1 pIutic ~ n-diluted annbodylenzyme eo1ution (15 x 15 x 4.5 an)ht,ffior ] IO.lM Tris pH 9.5, O.lM 50.C wata' b8tbNaa, O.O5M ~)eolor deYdopmeut solutionTE buller
Wash and Develop CoIor[70 minUte5; men 10+ minutes of deveJopment)
Gende agitatioD by hand should accompany each wash (incubatioo),~cept for IM coIor tkwlopment iii step 11.
I. Uång gIoved hand. and for<q>l, mnove tbc mombnne &om tbc ~hybridization solution (Part B), and transfer it to a small plastic: coo- rtainer with 100 mi of wash buffel:. Incubate at room tempera ture for N ___L _L- ..1-- .S minuta. Cftr toUQI UIC DI-' mcmbr311e With
Y°Ul' bare banda. 0iIs from your skinmar be deposiced on the membrane
2. Pour off the fint wash, and replace it with 100 mi of fresh wash and can prevent the anribody/mzymebuffer. Incubate, with agitation, at room temperatUre for S minuta. conjupte from bindiq.
3. Pour off the seeond wash, and replace it with 100 mi of fresh wasb Do not discard the hybridization solu-buffe~ Incubate, with agitation, at 50.C for S minUteL noa; coUea it fot reuel
4. Pour off the third wash, and add 100 mi of buffer 1. Incubate, withagitation, at room temperatUre for S minuta. ~S. Pour off buffer 1, and add 100 mi of buffer 2. Incubate, with agita-tion, at room temperature for 15 minuta.
6. Pour off buff er 2, and add 40 mi of the antibody/enzyme solution. ~Incubate, with agitation, at room temperature foc 15 minuta. .
7. Pour off the antibody/enzyme solution, and add 100 mi of buffer 1.Incubate, with agitation, at room temperature for S minUte8.
8. Pour off buffer 1, and replace it with 100 mi of fresb buffer 1. Incu-bate, with agitation, at room tempelatUre for S minUte8.
9. Pour off buffer 1, and add 100 mi of buffer 3.lncubate, with agita-tion, at room tempetature for S minUU:L .' -
10. Transfer the membrane to a dean~ . . disb or other suit- . ... " ~,.., "
.bl . r~ ".LI~ -.r:.. ',t'a e ~tama:. rP1 , u,
11. Add ~~ of freshly diluted eolor devdopment solution to tbe mem- '\ ~ =-~ Jbrane, and incubate at room temperature iii the dark. until the poIi- ~~-~
tiye colonies becomc dearfy visible (for 10 minuteI tO 1 bour). Malt.. If h h b ' dO' . . . L' I_-h th 1.-' .. d l !J- D . I t e y rllZ:1tlonslgna Js~amt."olW'sure t at e memurane IS pos'bone ca ony Suu: up. o not aB't4U devdopmmt can be atended
t'...
,~
~r /Ncver touch the nylon mcmbrane withyour bare handa. 0iJs from your skinmaJ' be deposited on the membraneand can prevent the antibody/mzymeconjugate from bindins-
Do not discard the hybridizarion solu-tion; col1ect it for tcUlel
~~"~.~~7
If the hybridizarion signal is faint. colordevdopment can be eXtendedovemight. However, ovcrincubationwill cause the entire membrane to bcgintO rum hmwn
260 ~16C..................................................................................................
The final wa,h in TE buffer stops thecoloc dcvelopment and prcvents funherdarkening of the background.
12. When the signals reach an acceptable levet, pour oH the developmentsolution, and add 20 mI of TE buffer. Incubate, with occasional ap-tatian, at room tempelature for a minimum of 30 minUteI.
13. Air dry the membrane on a piece of dem filter paper, and store it inthe dark.
RESULTS AND DISCUSSIONExamine your hybridiud replica membrane. The dark purplish-brown .ig-Dals result from the detection of the immune/digoxigenin complex andidentify transformants containing the 784-bp BamI-WHindIII Ä fragment.Compare your result to the ideal membrane bdow.
(
('
(,
\..
Ideal Hembrane
Use the registration marks to orient the replica membrane with its origi-nal UO or 1100 plate. (RemembeC that these two membranes are minorimages of each other.) Note the locations of colonies on the original mem-brane plate tbat correspond to hybridization signals on the replica mem-brane. Ohm, positive-reacting clones can be eaaily identified by compar-ing the pattem of hybridization signals on the replica membrane to thepattem of transformants on the original,UO or 1100 plate. If this provesdjffjcuJt, use a fdt-tip marker to transfer the patterD of hybridization sig-nals (including resistration marks) from the replica membrane to the lid ofa petri dish. The lid i. then placed over the originaJ plate and the registra-tion DW'lcs are aligned, thua simplifying the identification of positiveclones.
Remember that all colonies containins plasmids with any tyIM of Äimen are white. However, those with insetts other than 784 bp do notreaet with the probe and should not be visible on the hybridized mem-brane. Faint signals will become visible from negative white colonies wbcnthe eolor is overdeveloped, but these can be readily distinguished from thedarker signals of positive eolonics.
CoIony ~ of 1 Lb'8y 2 6 I
1. Fill in the matrix below with the foUowina:- 'T"L.- _n_L.- ~t ~:':n- &...&."':"":._':~- a:__I. ~.. "~n. ___1:-.. .ou.. u ~. ~.o 1'-'-'" "/~"--~" ~'e-- ~.. I~- ."1'-
membrane (I Positive)- . -. - . --- ----
D. ! DC: -ro-raJ numDcr 01 wnuc ~OIOIUC. UU UlIC Inallter ~U ur L.~UU
plate (' White).c. The percmtage of positive-reactina white colonies (% Positive).d. The number of elonable BamHllHindIII Å fragments (I Frag-
JDeDtI ) .e. The predicted percentage of positive-reacring white colonies COQ-
taining the 784-bp fragment, if each Å fragment is eloned with1 ~._/~7J1'" Iw.'.\
~~
-- o_, ,.-. -. -r'.f. Enter data for iteml a-< from severa1 other students, and the dau
I .~ '00-_000-'-
,,~O ..~~~oo..o..oooo.."oo~o..o .000" r~ ooooo..o"o~oo~..o
. " , , " ,...,..,... ., , '.........
2. What facton influence the doning frequency of the 784-bp A frag-ment versus other donable fragments?
30 a. CalcuJare the meaD (average) size of all restriction hagments gen-erated by the initial BamHIlHi"dJD digest of A that was used toconstrUCt the genomic library in Laboratory 140
bo Using the average restrienon fragment size from step a, estimatethe numher of fragments generated by a BamHI/HindJD digest ofthe human genome (haploid. 3 billion bp).
c. Calculate the total numhet of fragments created when the humangenome is restricted into fragments averaging 50,000 bp and500,000 bp.
d. What are the advantagel of using larger restriction fragmentswhen constructing a genomic library of human DNA?
e. What rea gents and techniquel are employed to generate Iargerestriction hagmentl?
4. The human genome has a G + C content of 41 %. UIe this fac:t to esti-mate the cutting frequency of the restrictioa enzyma EooR!(GAATIq and Smal (CCCGGG).
S. The numhet of m::ombinant clonel needed (N) to detect a particuJarDNA sequence in a genomie library is caIcu1ated aa foUoWl
N - Io(I-P)
1n(1- f),where In is the naturallogarithm, P is the desired probability (levet ofconfidence) that the library containJ a specific recombinant of inter-est, and f is the average fraenon (or percent) of the genome in a singlerecombinant.
(8
2 6 2 Laboratorv 16C
Calculate the number of recombinant donel needed to have a 99%probability of identifying a single-<:opy sequence in a mammaliangenome (3 x 10' bp), using the following vector system8:a. plasmid - average inJert size, 10,000 bp
b. Å phage - average inJert size, 20,000 bp
c. cosmid - average inaert size, 40,000 bp
d. YAC - average insert size, 1,000,000 bp
6. Identifying a suitable probe is a major challenge when screening agene library. In $Ome c:ases, the gene product can be isolated from tia-sue and amino acid sequence obtained from a portion of the purifiedpr9tein. Using this data, it is possible to synthesize an oligonu- (
deotide probe corresponding to the codoDl that specify the knoWDsequence of amino adds. However, the degeneracy of the genetic:code presents a problem, because most amino adds are specified bymore than ODe codon. The brute force solution is to prepare a fami1yof oligonudeotides that correspond to all possible DNA codoDl. A ':marc streamlined approach is to select an amino acid sequence withthe 1ealt redundancy-avoiding amino adds such as leucine, whichhas six codona.
Referring to the codan table, select the amino acid sequence from thepartial protein sequence below that will generate the smallest fami1yof oligonucleotide probes, each 17 bases in lengtb.
Ala-Leu-Met-Phe-Asp-Glu-De- T~A1a-Ser-Asn-Glya. Calculate how many different oligonudeotides must be prepared
to ensure that ODe will perfectly match the target gene sequence.b. Screening a genomic library with a familJ of oligonudeotide
probes yields a number of candidate dones. How can you deter-mine which positive-reacting dones contain the bona. fide gene?
If: position 2nd position :Ini po8ItIon~'~ ~'~
,U C A G,..., " ' , , " , , ,.., ,., ,..."",..".., ,.., , ".,
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(3' end)
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Purification andIdentification of Å
Clones
Laboratory 16, .Colony Hybridization of the Å Library, - identifiacolonia that have been tranaformed with recombinant plasmida contain-ing the 784-bp Å fragment. However, hybridization alone cannot confirmthe structure or genotype of the recombinant plasmid, wlUch mar weUcontain several 1 restriction fragments. Furthermore, poIitive-reactingtramformants can actuaDy carry two or mOR different plasmida. Theobjective of this laboratet)' is te confinn the molecular genotypel of pla-mid DNA from two of the positive-reacting dones from Laboratory 16.
In Part A, "Plasmid Minipreparation of )JpBLU Recombinants,- pla-mid DNA is isolated from ovemight cuJtureI of two difkrent positive-reacrlng elona from the original UO or L1oo plate (Laboratoria 15-16).In Part B, ..Restriction AnaJysis of Purilied )JpBLU Recombinants, - sam-pla of the plasmids isolated in Part A and contral samplcs of Å and pBLUare incubated with BamHl and HindIIJ. The digeated sampla and samplaof uncut plasmida are coelectrophoresed in an agarose gel. Comigration ofa 784-bp fragment in the digested minipreps and the digested 1 contralconfinna the SUCCtSSfuJ eloning of the sequence of interest. Comparisen ofminiprep fragments with 1 ratriction fragmen~phll evaluation of therelative såts of uncut, supercoiled plasmi~providea evidence of thestructure, size, and numhet of plasmida present in each of the transformeddona.
~
ORVAL A B
180late Pla8mld DNA
Laboratory I 7/P8rt APlasmld Mlnlpreparadon of AJpBlU RecombIn8na
(
PRELAB NOTES
Review the prelab notes in Laboratory 6A,pAMP.-
For Further Information
The protocol presented here is based on the foUowing published methods:
Birnboim, H. C. and J. Doly. 1979. A rapid a1ka1ine cxtracrloa metbod for 8CI'eCD-
iDs recombinant plasmid DNA NNdeie Acids ~ch 7: 1513.Ish-Horowia, D. and J. F. Burb. 1981. Rapid and cfficient cosmid docUna-
N~ Add6 Raarch 9: 298'.~
tI~I.
(8
of"PlasmidMinipreparatioa
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:=.: 'c :i~.- ~ ~
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~
Doubl~ all centrifug~ times if you areusing 8 small microfuge mat gmc:ralnapproximatdy 2,000 )( I.
Accurate pipetting is essenriaJ to goodpJa5mid )field. The volumes of reagcntsare precise!y cajibratcd so ~t thesodium hydroxide added in step 6 willbe nc:utraJiz.ed by the aceric acid instep 8.
Do not overm;x. Excessive agitationshears the single-stranded DNA anddecreun plasmid yidel.
~ ö
~/nfYP Spi" ~'Vtifr(f ~it (SO)f- 'ftj ( deM n Gl .b e s kr,,' n-i t1j
PLASMID MINIPREPARATION OFA/pBLU RECOMBINANTS
Supplle. and Equlpment.R """""""""~--."'" ........... ....
l00-l,<>OOiLI micropipenor + tipe10-100111 miaopipenor + tip8
(optioaal)0.5-10-,.1 mic:ropipettor + tip8
l.5-mI tubabeabr of crusbed ia:beabr lm wutrIused tipe10%-bleach solution or disinfectaat(aucb .. LyIOI)
tWo E. coli! WpBLU ovcrnight cuJtura
glucOR/frislEDTA (GTE)SDSlsodium hydroxide (SDSlNaOH)potasaium acetatelaceåc acid (KOk)
iaopropanoI95 % etbanoITrialEDTA (fE)
disposabJe glcm:Ihair dr7ermicrofusepaper towdapermanent markertest robe nek
Isolate Plasmid DNA
(SO minutes)
1. Shake the cu1ture robes to resuspend the E. coli cdJa.., y _L_I _n- 1 r _I __L_- __":~L n :_:":_1- y _L_I --- _.L- ...11. J.. A..8uo;& u..u r ...~ Tu"" A..8~ u ...~ ......
the other tube "M2." Transfer 1,000 ~ of each ovemigbt cultureinto a separate l.5-ml tube.
,j. ~Jose me caps, ana place me tuoes m a DalanaQ conngurabon m amicrofuge rotor. Spin for 1 minute to pellet the ceUa.
4. Pour off the supematant from both tubes into a waste beaker forlater disinfectjon. Take care not to disturb the aU pellets. Invert thetubes, and touch the moutha of the tuba to a dean paper rowe!, towie][ oH aJ much aJ possible of the rf!maini"8 SUpem8taDt.
S. Add 100 of ;ce-co/d GTE solution to each tube. Reswpend tbepellets by pipetting the solution in and out several times. Hold tbetuba up to the light to verify tbat tbe swpension is homogeneousand that no visible dumps of ceUs remain..
6. Add 200 of SDSlNaOH solution to each tube. Cose the capl, andmix the solutions by rapidly inverting tbe tuba about live timea.
7. Stand the tuba on ice for S minutes. The suspension will become re1-: 1.. ...1_., --
8. Add 150 J.Ll of ;ce-cold KOAc solution to each tube. Oose the capa,and mix the solutions by rapidly inverting the tubes about live tima.A white precipitate will immediately appear.
9. Stand the tubes OD ice for S minuteI.
Puriflcation and Identiflcation of1 acnes 267"' n nn n n............................................................
10. Place the tubes in a balanced configuration in a microfuge rotor, andspin for S minutes to pellet tbe precipitate along tbe side of the robe.
11. Transfer 400 J1l of supematant from the Ml tube inta a dem 1.S-mItube labeled "Ml.. Transfer 400 J1l of supernatant from tbe M2tube into a dean 1.S-mI tube labeled 8M2.. Avoid pipetting the",.~cipitate, and wipe off any precipitate tbat dings ta tbe outside of theup, before expelling tbe supematant. Discard old tubes containiog
preåpita1e.12. Add 400 J..Ll of isopropanol to each robe of supematant. Clese the
caps, and mix the solutions by rapidly inverting the tubes about 6vetimes. Stand the tubes at room temperature for only 2 min"tG.
f..
13. Place the robes in a bala1u:ed configuration in a microfuge rotor, andspin for S minutes to pellet the nudeic acids. Align the robes in therotor so that the cap binges point outward. The nudeic acid residue,visible or not, will coUect under the binge during centrifugation.
14. Pour off the supernatant from both robes. Talte care not to disturbthe nucleic acid pellets. Wiclc off as much as possible of the remain-inB aleohol on a paper towe1.
15. Add 200 JLJ of 95% ethanol to each tube, and close the cap.. Flidethe tubes several times to wash the nueleic acid pellets.
Stor~ th~ DNA in ethanol at -20.C unnl you are ready to connn,".
16. Place the tubes in a balanad configuration in a microfuge rotol; andspin for 2-3 minutes to recollect the nucleic acid pellets.
17. Pour off the supernatant from both tubes. Talte care not to disturbthe nuc/eic acid pellets. Wick off as much as possible of the remam-iDs alcohol on apaper towel.
18. Der the nucleic acid pellets, using one of following methods:
a. Direct a stream of warm air from a hair dryer over the openings ofthe rubel for about 3 minutes. Be careful not to blow the pelletsout of the tubes.
,(8
orb. dose the caps, and puJse the robes in a microfuge to pool the
remaining ethanol. Carefully use a micropipettor to draw off theethanol. Let the pellets air dry at room temperature for 10 min-uta.
Do S[~P Il quiddy: O1;tkc SUl"l' thoJt th",microfuge will b: iml1lcdi.atdy avail-ilhlc fnr:-tt.'fl 13.111" isnpropanc>1 prd-.:n."tioJlly pn :iritarl'S nndek .lcidsrnpidJy; how~'vcr. proteins anJ oth~rl'l.'lIlI);lr compon.:na rcm;lilling in solu-tion will :lJ~ bc:~n to pr~dpit:ut: withriJ11(',
,
~
The p.:lld willlik,'ly ,\WC,U drhcr 310 ariny, t\:ardmp-sh.:1ped sm~ar or smallpartid\.'S on I hl.' hemom of c,lch tu~.Ho'o\"Cver. pdl,,'t si:r-c is not a valid pr~-di,,'tl)r of pl3Smid yidd or quaiity. Alaq;e pelk.'t iii composcd primarily ofRNA. snlt. and ,dlular do:hris carricdover from dl\.' original pr,,~ipiriltc; asmall ~1It't. or olle diffjcult to St't:.Ofrl'l mc.:ans a dc,mer pn:pararion.
Nudeic.: add pelll:fS .trl: not suluhk indh.mol and will !lut n:susp:nll duringwasbing.
2 6 8 laboratorv 17 A
Air bubbles east in the robe wall can bemist.aken for ethanot droplcD.
~
t
t
If you are using a O.5-10-pJnUcropipenor, set it to 7.S J&1. and pipettwice.
19. Be sure that the nucleic acid pellet is dry and that all ethanol huevaporated before proceeding on to step 20. Hold each robe up tothe light to confirm that no ethanol dropletl remain and that thenucleic acid pellet, if visible, appears white and flaky. If ethanol i8still evaporatin& you will detect an a1cohol odor by sniffing themouth of the tube.
20. Add 15 ~ of TE to each robe. Resuspend the pellets by scraping withthe pipet tip and vigorously pipetting in and out. Rinse down the sideof the robe several times, concentrating on the area where the pelletshould have formed during centrifugation (beneath the cap binge).Make sure that all DNA ha. diasolved and that no particles remain:n ..t.. ...:.. ,... ,..n ..t.. 4. ,..I..t.. ",I-r- ------------
21. Keep the two DNA/TE solutions separate. Do "ot pool them intoone tubel
Freeu the DNA/TE solutions at -20.C until you ~ read)' to continu&naw before usiIIg.
22. TaJte the time for proper cleanup:
a. Segregate for proper disposal culture robes and miaopipettor tipsthat have come into contact with Bo coli.
b. Disinfect the overnight cultures, the tips, and the supematantfrom step 4 with a 10%-bleach solution or disinfectant (such alLysol).
c. Wipe down the lab bench with soapr water, a 10%-bleach solu-tion,ordisinfectaot.
d.- Wash your hands before leaving the laboratory.
RESULTS AND DISCUSSION
See thc "Rcsults and Discussion- scction of Laboratory 6A, "PlasmidMinipreparation of pAMP, - for a detailed diacussion of the biochemiJtry
of the alJcAlinc lysia mcthod for plasmid purification.
I. Set Up R..trlctlon
ADO B-nDlg.ata
~. . . . ... . ::..I~: HaO
pBWBuf/RN888HaO
~~,.'u. .
';'- . .t'
. - .' 'M2.J. ~ .4- . :" l. ' . ,.
'. ~,.,.BufIRN8Ie. :HP .
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II. Ca.. 0.8% Ag.roM 081
. ~~.' .' ...i 't~~.:Y~;:(~.
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Laboratory 17/rWt.RestrIctIon AnaIysIa al Purtr.d YpBLU Recomb/nlna
.
~
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2 7 O L.aboratorv I 78
PRELAB NOTES
Review the prelab nates in laboratory 3, "DNA Restriction and Elec-ttophoresia,. and Laboratory 68, "Restriction Analysis of PurifiedpAMP. .
For Further Information
The protoco) presented here is based on the following published methods:
Hening. Ro B., H. M. Goodman, and H. W. Boyer. 1974. AnaJysia of &oR! &q-ments of DNA from lambdoid bec:teriopbages and other viruscs by agaroee-gel electrophoresis. Journal of Vtrology 14: 1235.
Sharp, P. A., B. Solden, and J. Sambrook. 1973. Detection of two restrictioaendoDuclease activities in Haemophihu parainflunrzM usins anaIyricaIaproee-dhidiwn bromide dectrophorais. Biochemistry 12: 3055.
."
RESTRICTION ANALYSIS OFPURIFED Ä/pBLU RECOMBINANTS
MI and M2 miniprep DNAIJ'E0.1 J1WI pBUJ rt(. f0.1 J1WI Å DNA
BamHYH indIIISx restriction buHerIRNuedistilIed water
loading d7C0.8% agaroee1x TrisIBoraWEDTA (TBE) bulfer1 J1&'mI ethidium bromide
(or ucontami1fllnOll:0.05 M KMnO.0.2.5 N Ha0.25 N NaOH
I. Set Up Restriction Dige
~,111
{or tl«ontaminatiort:0.05 M KMnO.0.25 N Ha0.25 N N.OH
I
(10 rninUte5; then 30-40 minutes of incubation)
1. Use a permantnt marker to label seven l.S-mI robes, as showa in thematrix below. Restriction rnctions will be performed in these tuba.
2. Use the matrix belowas a checklist white adding reagents to eachreaction. Rtad down each column, adding the same reagent to .11appropriate tubes. Vse a fresh pipet lip for each reagent.
8 , BamHII~.~ ~~~.&J ~.I ~ ~.~~ ~"'~ .Hlndlll . HJO.
....
~
(- a-Mt-M2-A+Mt+M2+B+
Spi
SJ&!
SJ&.!
SJII -3. Aher adding aU of the above reagents, close the tops of the rubes and
mix.
"'l-'tt"'f, II ~tJ ~ j, "tehul ~" !/b1 vlh1~ (lucj f"- Kti 4:' i ,
(,(~ ~~IttI~\I/. tll-J*fl<J~~ J/Je't1 ~I Ijt \ ,'t ~, ((,~ ~tt\ 1 ~
.~"p'~Ie:s. ~ Equ.I~~ ...........O.S-IO-JAl micropipettor + tipl
1.5-mI tubesaJuminwn foi1bcaker for agaroeebeaJcer for wasteluscd tiplcamera and film (optionaJ)dispoa ble gIoftIelectropboresi8 box
maskinS tapemicrofuge (optional)Puafilm or waxed paper (optionaI)permanent marker
plutic wrap (optionaI)power au ppIytest tube nektramiUuminator3"-C water bath60.C wafer batb (for agaroee)
~
Refer to Laborator)' 3, "DNA h8tric-tion and EI«trophorcsis." for det:liJedinstructiona on setting up digcsa.
~
D I gests
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2 7 2 l.aboratory 178
&dDo not overincubt'lte: During a longerincubarion, the DNasn in the miniprepmar de<uade the plasnUd DNA.
Refer tO Laboratory J, "DNA Restric-rion and EJecrrophornis," for detailcdinstrucrlons on casrins and toading FL
:e them for4. Place the reactions in a 378C water bath, and incubat30-40 minutes onJy.
Following ;ncubation, {rene the reaaions af -20.C until you are read,to cont;nue. Thaw the read;ons before continuing on to Seaion III,step 1.
II. Cast 0.8% Agarose Gel
(15 minutes)I1. Carefully pour agarose solution into the ge1<asting ttay to fill it to a
depth of about S mm. The gel should cover about one-third theheight of the comb teeth.
2. Alter the agarose solidifies, place the gel<asting ttay into the elec-ttophoresil box and set up for electrophoraia.
CoveT the electrophoresis bo:c. and StIVe the gel untiI you are ,eady tocontmue. The gel wiU ,emain in goad et:mditiort for sweral åays if il iscompletely submerged in buffa-.
III. Load Gel and Electrophorese
(10 minutea; then 3~4S minUte8 of electrophoresia)1. Remove the restrictiOD digestl from the 3,.C water bath.2. Add 1 ~ of loading dye to each reaction robe. CIose the tops and
mix.
3. Load 10 ~ from each reactioD robe inta a separate weil in the gel, asshown in the diagram below. Use a fresh tip for each reactioa.
4. Electrophorese at 50-125 volts until the bromophenol blue bandshave moved 50-60 mm from the wells.
S. Tum off the power supply, remove the casting tray from the dec-trophoresis box, and transfer the gd to a disposable weigh boat (orother shallow rray) for staiJuns.-
The gel can be stored overnight, covered with small volume of buffer, ina staining trtry or in a lip-lock plastic bag, for viewing or photographingthe next day. However, over a longer period of time, the DNA will dif-fuse through the gel, and the bands will either become indistina or dis.
appear ennm,.
.I~t < Iv. Stain Gel with Ethidium Bromide, View, and
Photograph(15 minutes)
;"C:::.A:!~E
Review the seedon on "Ethldlum Bromide Stalnlng and Responslble Hand"""In Laboratory 3. War dlsposable g\oves when staIni"" Ylewlng, and pho-tographlng gel and during cleanup. Confine all stalnlng to a restrlcted sink ar-..
1. Ftood the gel with ethidiwn bromide solution (1 J1gImI), and allow itto stam for 5-10 minutes.
2. View the gel under an uJtravioJet transiJJuminator or other UV lightsource.{
\'~.
~
Ultravlolet light can damage your eyes. Never look dlrecdy at an unshlelded I.Nlight source wIthout eye protection. View only throuah a filter or safety glass.that absorb the hannful wavelengths.
3. Make an exposure of 1-2 seconds with the camera set at 08.Develop the print for the recommended time (approximately 4S see-onds at room temperature).
4. Take the time for proper cIeanup:
a. Wipe down the camera, the transillwninator, and the stalningarea.
.. . +
}' .fThe 7S4-bp Ä. fr:tgmcnt migrates justbehind the bt'nmophenol blue marker.Takc (arr lIot to r"n it aIr of thc (",d oftbl: gd. Howc\'er, if time allows, dec-trophorese until rhc bromophcnol hluebands m'ilr the l.'11d of the gel. This.llIows maximum sep.uation of uncutplasmids, whkh is import:\nt for diffcr-entiaring II large "supc:rplasmid" fromII double rmnsform;1rion of two smalJerplasmids.
Staining may be: p:rform~ by nninstrueror in :1 controUcd area, whenstudents ;tre nor present.
~ ~B:lDd inrc:nsity and conrrasr incn.'3SCdramarici1l1y if rh&." gc:l is dorainc...d for15-30 minute:\ in r.,p VI'ater. More sim-pir. rinse: and Jrain the Sds. stack thesraining rrays, cClver rhc: top ReI. and letir St'( ovcmight at room tc:mp..'t3tUre.
!D
27.. l.aboratory 178u_..............................................................................................................
b. Dccontaminate the gel and any staining solution that will not bereused.
c. Wash your hands beforc leaving the laboratory.
RESULTS AND DISCUSSION
Refer to the "Results and DiscussionW section of Laboratory 6B, "Restric-tion Analysia of Purified pAMP,w for more about interpreting miniprcp~els and plasmid conformation.
Examine the photograph of your stained gel, and determine whicb lana:ontain controi DNAs (pBLU and l) and which contain miniprepa MI andW. Even if you .have not foUowed the prescribed loading order, theniniprcp lanea can be distinguished by the foUowing charaaeristica:t A baclcground "smearW of degraded and partiaUy digested chromoso- I
mal and plasmid DNAt Undissolved material and DNA of high molccular weight trapped at the
&ont edge of the sample weUI A "eloudw of RNA of low molecular weight at a position corresponding
to 100-200 bpI The presence of high-m01ecular-weight bands of uncut plasmid in lanes
of digested miniprep DNA
Remember this when you are considering the possible .t:ructures of the'ccombined plasmids in MI and M2:
. Every replicating plasmid must have an origin of replication. Rccombi-nant plasmids with morc than one origin also replicate normaUy.
I Adjacent restriction fragments in a rccombinant plasmid must be lig-ated at like restriction sites: BamHI to BamHI and Hindm to HindIn.RccaJJ that, in Laboratory 10, "Purification and Identification ofRccombinant Plasmid DNA,w rccombinant plasmids were ligated hornrestriction fragments with one BamHI end and ODe HindIU end.Because of this, esch recombinant plasmid had to be compoaed of aneven number of fragments. The situation il morc complicated in thislaboratory, however. The BamHIlHindID digest of l in Part A creatednumerous restriction fragments; some have two BamHI ends or twoHindm ends. These can combine with BamHIlHindm restriction frag-ments to form plasmids composed of odd numbers of &agmenta.Repeated copies of the same restriction fragment cannat exist adjacentto one another; that il, ther must alternate with other fragments. Adja-cent duplicate fragments form "inverted repeats;W the sequences, one oneither side of the restriction site, are complementary along the entirelength of the duplicated fragment. Molccules with such inverted repeatscannot repticate properly. As the plasmid opens up to aUow access toDNA polymera se, the single-stranded regions on either side of therestriction site base pair to one another, forming a !arge "hairpin loop, w
which fouls repticatioo.
Use questions 1-10, to interpret esch pair of miniprep resuJtI (MI+!-I1d M2+1-).
1. Examine the photograpb of your stained gel. Campare your gel tothe ideal gel on the next page, and la bel the size of the fragments ineach Iaoe.
RESULTS
(
(.
e,
(
Use questions 1-10, to interpret each pair ofand M2+1-).
M1-B-
SlFERCOILEDpBW
....-
~
iI
IdeaJ Gel
2. Label the band in the rot pBLU lane (8+) .5,400 bp.. Then label thenine bands mat appear in the cut ~ laDe (~+).It is easiest to start withthe smallest fragment at the bottom of the gel and then progreutoward the sample weil. Remember that you must account for 13BamHJ/Hindmrestriction fragments. Notice that several thickbands are actually composed of multiple fragments, and the smaUeltfragment (125 bp) either runa off of the end of the gel or is too faintto be seen. A 14th fragment of 9,866 bp arisea from digesting circu-lar ~ molecu1es joined at the eos sita.
3. Label the rype(l) of restriction sitta at the enda of each Å fragment(Baml-ll-HindIII, Baml-ll-BamHl, or HindIII-HindIIl).
4. The cut miniprep tant!! (M + ) providn informatiO1l about the numbnand types of restrictiO1l fragments in th. construa. Every digestedminiprep must contain the S,400-bp pBLU backbone with the amp'gene and the 784-bp Å fragment that reaets with the hybridizationprobc.I. Locate these band. by comparins the rot miniprep laDes with the
cut pBLU and Å laDa.
b. It i. po5sibJe to lind miniprep. that do not conuin the 784-bp Åfragment. This resuJtI from beginning the ovemight cuJture with .nonreacting colony from the UO or LtOO original pJate.lnrxac:ralignment with the hybridized membrane makes it easy to mistak-enJy seJect a coJony Jocated very cIose to a positive-reacting done,especialJy if the master plate contains numerous, dosely spacedA_I__:------
S. Now, look for evidenee of other bands in the cut miniprep lanes. Ifno additionai fragments are visible, the molecule i. then termed a"simple recombinant8 of 6,184 bp. (This is MI of the ideal gelaOOve.)
M1+M2- M2+1+ B+
5.400 BP
7&4 BP
2 7 6 Laboratorv 178
6. Other than the simple recombinant, constructs are typically seen tocontain ODe additional ~ fragment, which can be cither
a. a three-fragment .superplasmidw composed of the pBLU back.bane, the 784-bp ~ fragment, and an additional ~ fragment withtwo BamHI enda or two HindJ]J enda. (Thia is M2 of the ideal gelon the previous page.)
}" .. I'nn...fr ~f' cn.-rnl..crnitl in ,.,}"irh nn~ nI' th~ th..- fr!lam~n"-- - --- --., p-,. --, - - ,..-------
above is repeated.
~ ~ ..I~..I.'- ._A_.t~_~..:~- ~t - .:__1- -_1.:_-- I_UIII 1.-"""--. - -- - r-- n 'r--- ----bane plUJ the 784-bp fragment) and another recombinant com-posed of pBLU plus the other visible &asment. In this situation.the additional fragmmt must haw OM BamHI end and OMHindIII mJ.
..-,. UIIC UU: ~ UI uu: '" .u~Ut ~UW ~ .,..~ U8 - U. ~
typcI of construcll: .
left end - BamHI 5,505 bpBamHl- BamHI 16,841 bpBamHl- HindIII 784 bpHindIIl- HindIII 2,027 bpHindIII - HindID 2,322 bpHindIII - &mHl 493 bpBamHl-BamHl 6,527 bp
BamHl- HindIII 2,396 bpHindID - HindID 564 bpHindIII - HindIII 125 bpHindIIl- BamHl 4,148 bp
BamHl- HindID 2,409 bpHindID - right end 4,361 bpa. three-&agment superplasmida - (
b. four-&agment superplasmida
c. two-&agment plasmids, other man the simple recombinant
8. The uncut miniprep lanes (M-) provide information about the over-aU size of the construct. Compare the M- (uncut miniprep) Jane with (the B- (uncut pBLU) laDe. Remember that the uncut plasmid canassume any of several conformationa, but the fastest-moving form il_u !I_..I5Upcn;Ouc:u.a. Locate the band that has migrated farthest in the B- lane; this is
tbc supercOile<l tarm ot pBLU.
b. Now examine the band famest down the M-Iane. If this band isonly slightly highu on the gel, man the molecuJe is most likelyasimple recombinant containing the 784-bp fragment.
c. The presente of supercoiled uncut miniprep DNA higher on thegel indicates alarger construct. Distinguishing berween doubletransformations and superplasmida can be difficuJt, because samerwo-fragment CODStructs are largu than othu three- or four-frag-ment constructs. Examining the type of enda on the additionaI
i
I
l.
fragment(s) in the M+ lane can orten narrow the range of possibleconstruct5, howeva:.
9. When bacteria are transformed using two different plasmids, one ofthe two may be preferentialJy replicared within the host cell. Overgenerations, the copy numbers of the two plasmids become increu-ingJy different. Thus, in double transformations showing four differ-ent fragments, one pair of fragments can be fainter than the otherpair.
10. Based on your above evaluation, make scale restriction maps of yourMI and M2 plasmids.
1
I.
.If
.FOR FURTHER RESEARCH
Analyze several transformants to ascertain whether the live clonable Åfragments with ont BamHl end and one HindID end are represented withequal frequency in the two-fragment COnstructl.
1. Start ovemight cultures from several white colonies picked from anUO or LtOO plate.
~
2. Prepare plasmid DNA from each ovemight culture, using the alJca-line lysis method.
3. Restriction anaJyze the purified plasmids with BamlD and H;ndID,using appropriare controIs.
,~
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