illustra nucleon genomic dna extraction kits · 2017-09-20 · flow diagram showing the principle...
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GE Healthcare
illustraNucleon GenomicDNA Extraction Kits
Product Booklet
Codes: RPN8501RPN8502RPN8509RPN8512
Contents1. Components of the system 3
2. Safety warnings and precautions 4
3. Storage and stability 5
4. Description 6
5. Flow diagram showing the principle steps in the
nucleon protocol 8
6. Critical parameters 9
7. Additional equipment and solutions required 107.1 Equipment 107.2 Solutions 10
8. Protocols 118.1 Nucleon BACC1, for 0.05-1.0 ml blood volumesor 1 to 3 x 106 cultured cells 11
8.2 Nucleon BACC2/3, for 3-10 ml blood volumesor 3 to 10 x 106 cultured cells 15
8.3 Nucleon HT for hard tissue 20
8.4 Nucleon HT for paraffin embedded tissue 24
9. Troubleshooting guide 28
10. Additional information 3610.1 Calculation of centrifugal force 3610.2 Additional reagent preparation 36
11. References 37
12. DNA extraction products 38
13. Legal Section 39
2
1. Components of the system
1. Nucleon kits for the extraction of genomic DNA from blood andanimal cell cultures (illustraTM Nucleon BACC, product code RPN8501/ RPN8502 / RPN8512) consist of:
Product Code Component Nucleon Nucleon Nucleon
BACC1 BACC2 BACC3RPN8501 RPN8502 RPN8512
Reagent A 220 ml 420 ml 510 ml(4 x concentrate)
Reagent B 18 ml 110 ml 110 ml
5 M Sodiumperchlorate 6 ml 26 ml 26 ml
Nucleon resin 8 ml 16 ml 16 ml
2. Nucleon kits for the extraction of genomic DNA from hardtissue (illustra Nucleon HT, product code RPN8509) consist of:
Component
Reagent B 18 ml
5 M Sodium perchlorate 6 ml
Nucleon resin 8 ml
Proteinase K 10 mg
3
2. Safety warnings and precautionsWarning: For research use only.Not recommended or intended for diagnosis of disease in humans oranimals. Do not use internally or externally in humans or animals.
All chemicals should be considered as potentially hazardous. Wetherefore recommend that this product is handled only by thosepersons who have been trained in laboratory techniques and that itis used in accordance with the principles of good laboratorypractice. Wear suitable protective clothing such as laboratoryoveralls, safety glasses and gloves. Care should be taken to avoidcontact with skin or eyes. In the case of contact with skin or eyeswash immediately with water. See material safety data sheet(s)and/or safety statement(s) for specific advice.
Note that the protocol requires the use of:Chloroform: carcinogen, Cat 3, harmful, irritant.Ethanol: highly flammable.Xylene: harmful, irritant.Concn hydrochloric acid: corrosive.Sodium hydroxide: corrosive, irritant.
Please follow the manufacturer’s Safety Data Sheet relating to thesafe handling and use of these reagents.
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3. Storage and stabilityStorageStore at room temperature, 15–30˚C. Proteinase K should be storedat 2–8˚C.
StabilityThe Nucleon kit components are stable for up to 18 months, 3months once opened, when stored under the recommendedconditions. Performance is consistent when stored under therecommended conditions using the recommended procedures.
ExpiryFor expiry date please refer to outer packaging label.
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4. DescriptionNucleonTM resin contained within the Nucleon genomic extractionkits was first developed by Professor Brian Caddy at StrathclydeUniversity, for use in extracting DNA from difficult forensic samples.The protocols and Nucleon resin have been further developed byscientists within Tepnel Life Sciences and by a number of academiccollaborators.
The systems supplied by GE Healthcare are designed to give highyields of pure DNA from blood, cultured cells, hard tissue or paraffinsections. The procedures have been optimized to give maximumrecoveries of high molecular weight DNA using a low shearingprotocol. The unique system employing the Nucleon resin removesprotein effectively without the use of phenol.
Typical yields and purities are shown in Table 1. The size of the DNArecovered from these tissues ranges from 23 to 250 kbp, asdetermined by pulse field gel electrophoresis. The combination ofpurity and high molecular weight makes the recovered DNA suitablefor a variety of molecular biology applications.
Table 1: Typical yields and purities.
Sample Yield mean purityμg DNA/mg tissue A 260/280
NucleonBACC1/2/3 Blood (10 ml) 370–440 1.8
HeLa cells (106 cells) 12 1.8Nucleon HT Mouse tail per cm* 70–200 1.9
Xiphisternum 1.6–1.9 1.6
* yield will vary depending on the section of tail used. 1 cm of tailequals approximately 60 mg of tissue.
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Nucleon BACC for blood and cell cultures.(see flow diagram on page 8)
Nucleon blood and cell culture (BACC) extraction kits are available in 3formats. Please note that the BACC2 kit is supplied with sufficientvolume of the non-proprietory Reagent A for 10 preparations from10 ml of blood or 3 x 106 to 1 x 107 cells.Reagent A preparation can be found in the additional informationsection of the protocol booklet.
illustra Nucleon BACC1for 50 preparations from 1 ml of whole blood or 1 to 3 x 106
cultured cells.
illustra Nucleon BACC2for 50 preparations from 10 ml of whole blood or 3 to 106 to 1 x 107
cultured cells.
illustra Nucleon BACC3for 50 preparations from 10 ml of whole blood.
illustra Nucleon HT for hard tissue and paraffin sections(see flow diagram on page 8)
Nucleon HT protocols have been developed for those tissues whichdo not homogenize easily in lysis buffer and require digestion withproteinase K. The kit has sufficient reagents for 50, 25 mgpreparations or 50 paraffin sections.
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5. Flow diagram showing the principlesteps in the nucleon protocol
Hard Tissue& Paraffin Section
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DNA - containingupper aqueous phase
Semi-solid stratum ofNucleon Resin with
bound proteins
Protein-containingorganic phase
Chophard
tissue
De-waxingof paraffin
sections
Proteinase K
BloodCell lysis
Deproteinisation withsodium perchlorate
Extraction withchloroform treatment
nucleon resin
DNA recovery
DNA washing
6. Critical parametersIt is strongly advised that an aseptic technique be used inconjunction with the described protocol, particularly whenhandling Reagent A. Once opened store at 2–8˚C.
Proteinase K is supplied as a powder which should be storedat 2–8˚C. Before use dissolve in 1 ml of sterile water. Store thesolution at 2-8˚C for up to three months.
Reagent A in the Nucleon BACC3 kits is supplied as a 4xconcentrate. Prior to first use it should be diluted four foldwith deionized water and autoclaved in suitable aliquots.
It has been reported that heparin can bind to DNA duringextraction. If present in the final DNA solution it can causeinterference with PCR* techniques (1). The effect of heparinmay be counteracted in several ways (2,3).
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7. Additional equipment and solutions
Microcentrifuge
Bench top centrifuge
1.5 ml polypropylene microcentrifugation tubes
12 mm diameter polypropylene screw capped tubes
Assorted range of high precision pipettes
Water bath
Rotary mixer
Homogenization tube
7.1 Equipment•
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•
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•
•
•
•
Chloroform, AnalaRTM grade or similar
Ethanol, AnalaR grade or similar
Xylene, AnalaR grade or similar
RNase solution, 50 μg/ml in sterile water
Dry ice
TE buffer
1.21 g TrizmaTM base0.372 g Ethylenediaminetetra-acetic acid, sodium saltAdd approximately 800 ml of distilled water. Mix to dissolve. Adjust topH 8.0 with concentrated hydrochloric acid. Make up to a final volumeof 1000 ml. Autoclave in suitable aliquots.Store up to 3 months at room temperature.
7.2 Solutions•
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8. Protocols8.1 BACC1 for small blood volumes (50 μl-1.0 ml)and 1 x 106 to 3 x 106 cultured cells.The protocol requires the use of cold 70%(v/v) and absolute ethanol.
Protocol
Cell Preparation
Notes
Collect the blood(50 μl–1.0 ml) in sodiumEDTA tubes.OrCollect the cultured cellsby centrifuging at 600 g for5 minutes at 4˚C. Discardthe supernatant withoutdisturbing the pellet.
Using an aseptic procedureadd 4 times the volume ofReagent A to the bloodsample. Rotary mix for 4minutes at room temperature.Centrifuge at 1300 g for 5
1.
2.
In order to minimize damageto DNA in collected bloodsamples, blood which is storedat 4˚C should be extractedwithin 24 hours of collection.Heparinized and citrated bloodis also suitable. Heparin mayinterfere with subsequentprocedures, see criticalparameters page 9.Harvested cells from buccalswabs/spatulas in appropriatemedia for example isotonicsaline, sucrose etc. and buffycoat preparations can beprocessed using the BACCprotocols.
Due to the high sucrosecontent, Reagent A could becontaminated if aseptictechnique is not used whendispensing.
1.
2.Cell Lysis
minutes. Discard thesupernatant.OrResuspend cells in 1.0 mlReagent A and leave on ice for5 minutes. Centrifuge at1300 g for 5 minutes anddiscard the supernatant.
To the pellet add 350 μl ofReagent B. Vortex briefly toresuspend the pellet.Transfer the suspension to a1.5 ml microcentrifuge tube.
Following resuspension andtransfer to a clean tube add2.5 μl of a 50 μg/ml RNasesolution. Incubate the tube ina water bath at 37˚C for 30minutes.
Add 100 μl of sodiumperchlorate solution. Mix byhand, inverting the cappedtube at least 7 times.
3.
4.
5.
Ensure that Reagent B iscompletely dissolved. This maybe achieved using gentle heat.Incubating the samples at37˚C for 10 minutes can alsohelp to resuspend the pellets.
Extracted DNA from culturedcells may contain smallamounts of RNA. If RNA freeDNA is required, an RNasedigestion step should beincluded. RNase should bemade up in water and boiledfor 10 minutes to inactivateany contaminating DNase.
3.
4.
12
Protocol Notes
RNase treatment (optional)
Deproteinisation
DNA extractionAdd 600 μl of chloroform (notsupplied). Mix by hand,inverting the capped tubeat least 7 times.
Without remixing the phasesadd 150 μl of Nucleon resin.Centrifuge at 350 g for1 minute.
Without disturbing theNucleon resin layer (brown incolor), transfer the upperphase (approximately 450 μl)to a clean 1.5 mlMicrocentrifuge tube.
6.
7.
8.
13
7.
8.
A spin speed of 350 gcorresponds to 2000 rpm in anEppendorf TM 5415 Microfuge.Check the manualaccompanying your machineor refer to the additionalinformation section page36. Speeds higher that thosegiven may cause the resin tospin to the bottom of the tube.
The resin layer should not bedisturbed in order to minimizecontamination from theprotein interface. A whiteprotein layer may also beassociated with the resin. Thislayer must not be disturbed.If any resin has been carriedover, centrifuge briefly at aminimum of 1300 g to pelletthe resin, and then transfer toa clean tube. The resin, ifcarried over, will not interferewith subsequent processing.
Protocol Notes
DNA precipitation
Add 2 volumes (900 μl) of coldabsolute ethanol. Mix byinversion until the precipitateappears.
Centrifuge at top speed(minimum 4000 g) for 5minutes to pellet the DNA.Discard the supernatant.
Add 1 ml cold 70% (v/v)ethanol, mix several times byinversion. Re-centrifuge anddiscard the supernatant. Thisstep can be repeated ifnecessary.
Air dry the pellet for 10minutes, ensuring that all theethanol has been removed.Re-dissolve the DNA in anappropriate volume of wateror TE buffer (e.g. 50–250 μl).The DNA should re-dissolvewithin 2 hours when using arotary mixer.
9.
10.
11.
12.
14
Protocol Notes
DNA washing
15
Protocol
Cell Preparation
Notes
8.2 BACC2/3 for 3 - 10 ml blood and3 x 106 to 1 x 107 culture cells.Note: Reagent A in the BACC3 kit is supplied as a 4x concentrate.Prior to first use it should be diluted four fold with deionized waterand autoclaved, at 121˚C 15 psi for 15 minutes, in suitable aliquots.The protocol requires the use of cold 70%(v/v) and absolute ethanol.
Collect the blood in sodiumEDTA tubes.OrCollect the cultured cells bycentrifuging at 600 g for5 minutes at 4˚C. Discardthe supernatant withoutdisturbing the pellet.
Using an aseptic procedureadd 4 times the volume ofReagent A to the bloodsample. Rotary mix for 4 minutes at room
1.
2.
In order to minimize damageto DNA in collected bloodsamples, blood which is storedat 4˚C should be extractedwithin 24 hours of collection.Heparinized and citrated bloodis also suitable. Heparin mayinterfere with subsequentprocedures, see criticalparameters page 9.Harvested cells from Buccalswabs/spatulas in appropriatemedia for example isotonicsaline, sucrose etc. and buffycoat preparations can beprocessed using the BACCprotocols.
Due to the high sucrosecontent, Reagent A could becontaminated if aseptictechnique is not used whendispensing.
1.
2.Cell Lysis
temperature. Centrifugeat 1300 g for 5 minutes.Discard the supernatant.OrResuspend cells in 1.0 mlReagent A and leave on icefor 5 minutes. Centrifuge at1300 g for 5 minutes anddiscard the supernatant.
To the pellet add 2 ml ofReagent B. Vortex brieflyto resuspend the pellet.Transfer the suspensionto a 15 ml screw cappedpropropylene centrifuge tube.
Following resuspension andtransfer to a clean tube add15 μl of a 50 μg/ml RNasesolution. Incubate the tubein a water bath at 37˚Cfor 30 minutes.
Add 500 μl of sodium
3.
4.
5.
Ensure that Reagent B iscompletely dissolved. This maybe achieved using gentle heat.The internal diameter of thetube should not exceed 12 mm.Incubating the samples at37˚C for 10 minutes can alsohelp to resuspend the pellets.
Extracted DNA from culturedcells may contain smallamounts of RNA. If RNA freeDNA is required, an RNasedigestion step should beincluded. RNase should bemade up in water and boiledfor 10 minutes to inactivateany contaminating DNase.
3.
4.
16
Protocol Notes
RNase treatment (optional)
Deproteinisation
perchlorate solution. Mix byhand, inverting the cappedtube at least 7 times.
Add 2 ml of chloroform(not supplied). Mix by hand,inverting the capped tubeat least 7 times.
Without remixing the phasesadd 300 μl of Nucleon resin.Centrifuge at 1300 g for3 minutes.
Holding the tube verticallywithout disturbing theNucleon resin layer (brownin color), transfer the upperphase (approximately 2.5 ml)to a clean tube of minimumvolume 7.5 ml.
6.
7.
8.
A spin speed of 1300 gcorresponds to 2400 rpm ina centrifuge with a swing-out bucket rotor radius190 mm. Check the manualaccompanying your machineor refer to the additionalinformation section page 36.Speeds higher than thosegiven may cause the resin tospin to the bottom of the tube.
The resin layer should not bedisturbed in order to minimizecontamination from theprotein interface. A whiteprotein layer may also beassociated with the resin andthis must be avoided. If anyresin has been carried over,centrifuge briefly at a
7.
8.
17
Protocol Notes
DNA extraction
DNA precipitation
Add 2 volumes of coldabsolute ethanol. Mix byinversion until theprecipitate appears.
Centrifuge at top speed(minimum 4000 g) for 5minutes to pellet the DNA.Discard the supernatant.
Add 2 ml cold 70% (v/v)ethanol, mix several timesby inversion. Re-centrifugeand discard the supernatant.This step can be repeated ifnecessary.
Air dry the pellet for 10minutes, ensuring that all theethanol has been removed.Re-dissolve the DNA in anappropriate volume of wateror TE buffer (e.g. 1.0–2.0 ml).
9.
10.
11.
12.
minimum of 1300 g to pelletthe resin, and then transfer toa clean tube. The resin, ifcarried over, will not interferewith subsuquent processing.
Precipitated DNA may behooked out at this stage usinga heat-sealed Pasteur pipette.This DNA does not require a70% ethanol wash and shouldbe placed directly into TE orsterile water.
9.
18
Protocol Notes
DNA washing
The DNA should re-dissolvewithin 2 hours when using arotary mixer.
19
Protocol Notes
20
Protocol
Tissue preparation and lysis
RNase treatment (optional)
Notes
8.3 Nucleon HT for DNA extraction from hardtissueNote: Proteinase K is supplied as a powder which should be stored at2–8˚C. Before use dissolve in 1 ml of sterile water. Store the solutionat 2–8˚C for up to three months.The protocol requires the use of cold 70%(v/v) and absolute ethanol.
Grind 25 mg of tissue on dryice or in liquid nitrogen to afine powder and transfer to a1.5 ml microcentrifuge tube.
Add 0.35 ml of Reagent B.
1.
2.
Add RNase solution to a finalconcentration of 400 ng/ml.Incubate the tube in a waterbath at 37˚C for 30 minutes.
Add 18 μl of the proteinase Ksolution and incubate at 50˚Cfor at least 3 hours (orovernight).
Centrifuge at 2000 g for
3.
4.
5.
For mouse tails it may benecessary to grind the tissuewith pestle and mortar afterfinely chopping.
Ensure that Reagent B iscompletely dissolved. This maybe achieved using gentle heat.
1.
2.
Extracted DNA from tissuemay contain small amounts ofRNA. If RNA-free DNA isrequired, an RNase digestionstep should be included. RNaseshould be made up in waterand boiled for 10 minutes toinactivate any contaminatingDNase.
3.
5 minutes. Remove thesupernatant and transferto a clean tube.
DeproteinisationAdd 100 μl of sodiumperchlorate solution. Mix byhand, inverting the cappedtube at least 7 times.
6.
21
DNA extractionAdd 600 μl of chloroform(not supplied). Mix by hand,inverting the capped tubeat least 7 times.
7.
Add 150 μl of Nucleon resinand without remixing thephases centrifuge at 350 gfor 1 minute.
8. A speed of 350 g correspondsto 2000 rpm in an Eppendorf5415 Microfuge. Check themanual accompanying yourmachine or refer to theadditional information sectionpage 36.
8.
DNA precipationWithout disturbing theNucleon resin layer (brownin color), transfer the upperphase to a clean tube.
9. The resin layer should not bedisturbed in order to minimizecontamination from theprotein interface. A whiteprotein layer may also beassociated with the resin.This layer must be avoided.The resin, if carried over, will
9.
Protocol Notes
Add 2 volumes of coldabsolute ethanol. Mix byinversion until theprecipitate appears.
Centrifuge at top speed(minimum 4000 g) for5 minutes to pellet the DNA.Discard the supernatant.
Add 1.0 ml cold 70% (v/v)ethanol, mix several timesby inversion. Re-centrifugeand discard the supernatant.This step can be repeated ifnecessary.
Air dry the pellet for 10minutes, ensuring that all theethanol has been removed.Re-dissolve the DNA in an
10.
11.
12.
13.
not interfere with subsequentprocessing. If any resin hasbeen carried over, centrifugebriefly at a minimum of 1300 gto pellet the resin, and thentransfer to a clean tube.
Precipitated DNA may behooked out at this stage usinga heat-sealed Pasteur pipette.This DNA does not require a70% ethanol wash and shouldbe placed directly into TE orsterile water.
10.
22
Protocol Notes
DNA washing
appropriate volume of wateror TE buffer. The DNA shouldre-dissolve within 2 hourswhen using a rotary mixer.
23
Protocol Notes
24
Protocol
Preparation of paraffin sections
Notes
8.4 Nucleon HT for DNA extraction fromparaffin sectionsNote: Proteinase K is supplied as a powder which should be stored at2–8˚C. Before use dissolve in 1 ml of sterile water. Store the solutionat 2–8˚C for up to three months.The protocol requires the use of cold 70%(v/v) and absolute ethanol.
Take one 20–30 micron sectionof tissue and place in a 1.5 mlmicrocentrifuge tube.
Cover the section in xylene.Incubate at 37˚C for 20minutes. Centrifuge at 1300 gfor 5 minutes and remove thexylene.
Incubate in xylene at roomtemperature for 2 minutes.Centrifuge at maximumspeed for 5 minutes andremove all the xylene.
Rehydrate the section bywashing consecutively in100% ethanol, 75(v/v)%ethanol, 50(v/v)% ethanol,25(v/v)% ethanol and finallywater. Centrifuge at maximumspeed for 1–3 minutesbetween each wash.
Remove the water from the
1.
2.
3.
4.
5.
Check the manualaccompanying your machineor refer to the additionalinformation section page 36.
Care should be taken at the25%(v/v) ethanol and waterstages as the material canbecome loose and difficultto pellet.
Ensure that Reagent B is
2.
4.
5.
pellet and add 0.35 ml ofReagent B.
Add 18 μl of the proteinase Ksolution and incubate at 55˚Covernight for maximum yield.
6.
completely dissolved. This maybe achieved using gentle heat.
A three hour incubation shouldprovide an adequate yield.
6.
25
DeproteinisationTo the 350 μl lysate add 100 μlof sodium perchlorate solution.Mix by hand, inverting thecapped tube at least 7 times.
7.
DNA extraction
Add 600 μl of chloroform(not supplied), Mix by hand,inverting the capped tubeat least 7 times.
8.
Add 150 μl of Nucleon resinand without re-mixing thephases centrifuge at 350 gfor 1 minute.
9. A speed of 350 g correspondsto 2000 rpm in an Eppendorf5415 Microfuge. Check themanual accompanying yourmachine or refer to theadditional informationsection page 36.
9.
Protocol Notes
DNA precipationWithout disturbing theNucleon resin layer (brownin color), transfer the
upper phase to a cleantube.
10. The resin layer should not bedisturbed in order to minimizecontamination from theprotein interface. A whiteprotein layer may also be
10.
Add 2 volumes of coldabsolute ethanol. Mix byinversion and leave at -20˚Cfor 1–2 hours to precipitatethe DNA.
11. 1 μl of 20 mg/ml glycogensolution may be added as acarrier if required.
11.
associated with the resin. Thislayer must be avoided. Theresin, if carried over, will notinterfere with subsequentprocessing. If any resin hasbeen carried over, centrifugebriefly at a minimum of 1300 gto pellet the resin, and thentransfer to a clean tube.
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Protocol Notes
DNA washingCentrifuge at top speed for15 minutes to pellet the DNA.Discard the supernatant.
Add 1.0 ml cold 70% (v/v)ethanol, mix several times byinversion. Re-centrifuge anddiscard the supernatant. Thisstep can be repeated ifnecessary.
Air dry the pellet for10 minutes, ensuring that allthe ethanol has been removed.Re-dissolve the DNA in an appropriate volume of water
12.
13.
14.
27
or TE buffer. (e.g. 100 μl). TheDNA should re-dissolve within2 hours when using a rotarymixer.
Protocol Notes
28
Problem Possible cause Remedy
9. Trouble shooting guide
Cell preparation forwhole blood (BACConly). Incompletelysis of red bloodcells resulting in awhite cell nucleipelletscontaminated withred cells aftertreatment withReagent A andsubsequentcentrifugation.
Cell preparation forwhole blood (BACConly).White cell nucleipellet has a hint ofred coloration.
1.
2.
Exceptionally highred cells counts inthe blood sample.OrWhite cellclumping aroundred cells therebypreventing red celllysis.
This may occur ifthe blood sampleis not as highquality as normalfor example as aresult of thecollection tube notbeing mixedor storedappropriately.
1.
2.
Repeat theReagent Atreatment as perthe protocol using1 ml Reagent A.
In most cases thiscoloration willdisappear uponcontinuation of theprotocol to thepoint of DNAwashing. Thereshould be no effectupon the suitabilityof the extractedDNA for furtherapplications. If thisis not the case,repeat the ReagentA treatment asper the protocol.
1.
2.
29
Cell preparation forwhole blood (BACConly).White cell nucleipellet fails to formor be retained atthe bottom of thetube.
Cell lysisIncomplete celllysis.
3.
4.
Insufficientcentrifugal force.
When the sampleis small (e.g. <5 mlblood), the pelletmay be small anddifficult tovisualize.
Pellets are morelikely to dislodgefrom round-bottomed tubesthan with conical-bottomed tubes.
Detergent inReagent B hascome out ofsolution.
3.1
3.2
3.3
4.1
Check thecalculation of gfrom rpm for yourrotor using theformula on page 36or from the rotormanufacturersmanual. If the gcalculation iscorrect, spin forlonger until thepellet forms.
Do not beconcerned. Proceedwith the protocolas normal.
Use conical-bottomed tubes orbe extra carefulwhen decantingthe supernatant.
Re-dissolve thedetergent byheating to 35˚Cand agitating.
3.1
3.2
3.3
4.1
Problem Possible cause Remedy
30
Cell lysisIncomplete lysis ofpellet stored inDMSO or isolatedfrom a Ficollgradient (for
5.
Too many cells inthe sample.
Pelleted material“clumps” often asa result of 4.2.above.
Contamination ofpellet with DMSOor Ficoll.
4.2
4.3
5.
Check the cellcount. If there aretoo many cells, splitthe sample asappropriate andproceed with theprotocol ensuringthat sufficientproportionalvolumes ofReagents A, B,sodium perchlorateand chloroform areused to preventoverloading of thesystem.
If “clumping” is theproblem, ensurethat the pellet isfully re suspendedto homogeneity inReagent B byvigorous vortexingor extendedincubation at 37˚Corroom temperature.
Wash pellet inphosphate bufferedsaline prior toadding Reagent B.
4.2
4.3
5.
Problem Possible cause Remedy
31
example BuffyCoat preparations).
DNA ExtractionThe brownNucleon resin failsto form a layer atthe interfacebetween thechloroform andaqueous phase.
6. The system isoverloaded due tothe presence oftoo many DNAcontaining cells.The resin does notclear the upperphase due to highviscosity causedby highconcentrations ofDNA.
The centrifugationspeed is incorrect.If too high, theresin may pellet atthe bottom of thetube.
6.1
6.2
Reduce the samplesize to fall withinthe recommendedrange for theprotocol in use.
Check thecalculation of gfrom rpm using theformula on page 36orfrom the rotormanufacturersmanual.Please note that if asmall amount ofthe resin does spinto the bottom ofthe tube(particularly withthe small volumeprotocols), this willnot affect thequality of the DNApreparation.
6.1
6.2
Problem Possible cause Remedy
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DNA precipitationSome Nucleonresin is carriedover into the DNApellet whichappears brown/redin color.
DNA precipitationA non-DNAinsoluble whiteprecipitate formson the addition ofethanol.
DNA washingThe DNA pellet willnot re-dissolve orre-dissolvesslowly.
7.
8.
9.
Tubes withinappropriatedimensions arebeing used.
Insufficient carehas been takenwhen removingthe upper phase orthe centrifugationafter thismanipulation hasnot beenperformed.
An excess ofethanol has beenadded.
The DNA pellet hasbeen excessivelydried.
The DNA is notpure.
6.3
7.
8.
9.1
9.2
The protocol hasbeen optimizedusing tubes withinternal diameterswhich ensureoptimal volumes ofNucleon resin toenable adequateprotein binding andbarrier formation atthe interface.
Follow the protocolas stated withadequate care.
Ensure that therecommendedvolumes of ethanolare not exceeded.
Follow therecommendeddrying conditions inthe protocol.
Please see 11below.
6.3
7.
8.
9.1
9.2
Problem Possible cause Remedy
33
DNA YieldLow Yield of DNA.
10.
The whiteprecipitatediscussed in 8above is beingmistaken for DNA.
Too few nucleatedcells present in thestarting sample.
Too manynucleated cellspresent in thestarting sample. Ifthis is the case,incomplete lysismay occur (see 4above).
9.3
10.1
10.2
Please see 8 above.
Check cell countprior to cell lysisstep to ensurethat the samplefalls within therecommendedrange.If a low yield isexpected due to anextremely smallsample size,recovery maybe enhanced byadding a carrierDNA (e.g.denatured herringsperm DNA) orglycogen (1 μl of20 mg/ml glycogenper 600 μl ethanol).
System isoverloaded,(see 4 above).
9.3
10.1
10.2
Problem Possible cause Remedy
34
Poor quality DNAThe A260/280ratio is too high ortoo low. The ratioshould fall in therange 1.7–1.9.
11.
Incomplete lysis(see 4 above).
Poor recovery ofDNA whenprecipitated withethanol.
Inaccurate UVmeasurement.
Loss of DNA-containing pelletafter discardingReagent A.
Low ratios usuallyindicate proteincontamination.
10.3
10.4
10.5
10.6
11.1
See 4 above.
Check thecalibration ofthe UVspectrophotometerusing a standardDNA solution.
See 10.1 above
Check thecalibration ofthe UVspectrophotometerusing a standardDNA solution
Take care whenremoving ReagentA. If in doubt, use apipette andcarefully drain theremaining ReagentA onto a tissue.
Avoid using samplesizes which exceedthe range for theprotocol beingused.
10.3
10.4.1
10.4.2
10.5
10.6
11.1
Problem Possible cause Remedy
35
The DNA isnot thoroughlyre-dissolved afterprecipitation.
11.2 Ensure that theDNA is fullydissolved afterwashing. AfterDNA pelletresuspension, leavethe samplesovernight at 4˚C.
11.2
Problem Possible cause Remedy
10. Additional information10.1 Calculation of centrifugal forceTo ensure that Nucleon protocols are universally applicable to allcentrifuges, centrifugal force is expressed as g rather than rpmvalues. To convert rpm to g please refer to the rotor manufacturersmanual. If this is not available use the formula illustrated below.
g = 1.12r (rpm/1000)2
rpm = 1000√(g/1.12r)r = maximum radius of the rotor in mm
10.2 Additional reagent preparationReagent A for illustra Nucleon BACC2 kits
10 mM Tris-HC1, 320 M sucrose, 5 mM MgCl2,1%(v/v) Triton X-100, pH 8.0
Combine reagents in 80% of the volume required. Mix to dissolve.Adjust pH to 8 using 40%(w/v) NaOH. Make up to volume and mixwell. Autoclave suitable aliquots at 121˚C 15 psi for 15 minutes.
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11. References1. BEUTLER, E. et al., Bio Techniques, 9 (2), p166, 1990.2. POLI, F. et al., PCR Methods and Applications, 2, pp356-358, 1993.3. TSAI, M. et al., American Journal of Pathology, 146 (2), pp335-343,
1995.
37
12. DNA extraction productsillustra Nucleon HT,for hard tissue, and paraffin sections,50 preparations of up to 25 mg per prep.
illustra Nucleon PhytoPureTM,for plant and fungal DNA extraction kit,50 preparations of 0.1 g.
illustra Nucleon PhytoPure,for plant and fungal DNA extraction kit,50 preparations of 1.0 g.
illustra Nucleon BACC1,for 50 preparations of 1 ml whole blood orcultured cells (1 to 3 x 106).
illustra Nucleon BACC2,for 50 preparations of 10 ml of whole blood orcultured cells (3 x 106 to 1 x 107).
illustra Nucleon BACC3,for 50 preparations of 10 ml of whole blood.
RPN8509
RPN8510
RPN8511
RPN8501
RPN8502
RPN8512
38
13. Legal SectionGE, imagination at work and GE monogram are trademarks ofGeneral Electric Company.
illustra is a trademark of GE Healthcare companies.
This product is sold under licensing arrangements with RocheMolecular Systems, F Hoffmann-La Roche Ltd and the Perkin-ElmerCorporation. Purchase of this product is accompanied by a limitedlicense to use it in the Polymerase Chain Reaction (PCR) process forresearch in conjunction with a thermal cycler whose use in theautomated performance of the PCR process is covered by theup-front license fee, either by payment to Perkin-Elmer or aspurchased, i.e. an authorized thermal cycler.
Nucleon and PhytoPure are trademarks of Tepnel Life Sciences plc.
All third party trademarks are the property of their respectiveowners.
© 1999-2007 General Electric Company - All rights reserved.Previously published 1999.
All goods and services are sold subject to the terms and conditionsof sale of the company within GE Healthcare which supplies them. Acopy of these terms and conditions is available on request.
Contact your GE Healthcare Representative for the most currentinformation and a copy of the terms and conditions.
http://www.gehealthcare.com/lifesciences
GE Healthcare UK Limited.Amersham Place, Little Chalfont,Buckinghamshire, HP7 9NAUK
39
For contact information for your local office,please visit: www.gelifesciences.com/contact
GE Healthcare UK Limited.Amersham Place,Little Chalfont, Buckinghamshire,HP7 9NA, UK
http://www.gehealthcare.com/lifesciences
RPN8501PL Rev B 11/2007
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