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PUBLISHED FOR THE LIFE SCIENTIST BY LIFE TECHNOLOGIES, INC. VOLUME 21 • NUMBER 3 • 1999 PUBLISHED FOR THE LIFE SCIENTIST BY LIFE TECHNOLOGIES, INC. VOLUME 21 • NUMBER 3 • 1999

Featured Papers:Transfection in 96–well Plates –58

Mammalian Cell Lines for Transfection – 62

Fluorescent DNA Ladders – 64

Helpful Tips for Custom Primers – 69

Some Basics of Cell Culture Media – 76

Featured Papers:Transfection in 96–well Plates –58

Mammalian Cell Lines for Transfection – 62

Fluorescent DNA Ladders – 64

Helpful Tips for Custom Primers – 69

Some Basics of Cell Culture Media – 76

c o n t e n t s

FOCUS®

TRANSFECTION

Transfection of Mammalian Cells in 96-Well Plateswith LIPOFECTAMINE™ 2000 Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Jean-Pierre Pichet and Valentina Ciccarone

Cationic Lipid Reagent Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61High Transfection Efficiency of Cloned Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Linda Roy, Sharon Cates, Kevin Schifferli, Jean-Pierre Pichet, Valentina Ciccarone,Shelley Bennett, and Pamela Hawley-Nelson

ELECTROPHORESIS

DNA Ladders for Fluorescent Fragment Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64Heather Jordan and Joseph Solus

PCR

One-Step RT-PCR to Detect Cytokine/Chemokine Induction in Macrophages . . . . . . . . . . . . . . . . . 66Monique Bongers, Ekke Liehl, and Johannes Barsig

Helpful Tips for Custom Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69Technical Services

AGRICULTURAL BIOTECHNOLOGY

AFLP™ Analysis of the Fruit Fly Ceratitis capitata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72Gino Corsini, Augusto Manubens, Manuel Lladser, Sergio Lobos, Daniela Seelenfreund, and Carlos Lobos

MOLECULAR BIOLOGY

Buffer Compatibility for Common Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74Joe Crouse, Teresa Myers, and Julie Brent

CELL CULTURE

How Basal Media Provide an Optimal Growth Environment for Cell Culture . . . . . . . . . . . . . . . . 76Kevin Grady

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F O C U S ( 1 9 9 9 ) V O L U M E 2 1 N U M B E R 3 57

FOCUS contains manuscripts describing novel techniques, improvements ofcommon techniques, simplified protocols, and troubleshooting.“Instructionsto Authors” are available on the Internet or from the editor:

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Purchase of Taq DNA Polymerase is accompanied by a limited license to use itin the Polymerase Chain Reaction (PCR) process for research and developmentin conjunction with a thermal cycler whose use in the automated performanceof the PCR process is covered by the up-front license fee, either by payment toPerkin-Elmer or as purchased, i.e., an authorized thermal cycler. This product issold under licensing arrangements with F. Hoffmann-La Roche Ltd., RocheMolecular Systems, Inc., and The Perkin-Elmer Corporation Inc.

99-142 Part No. 53077

FOCUS Editor:Dr. Doreen CupoLife Technologies, Inc.9800 Medical Center DriveRockville, MD 20849-6482(800) 828-6686 (301) 610-8000 outside the U.S.E-mail: [email protected]

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Contributing Editorial Reviewers:J.J. Lin, Dave Schuster

© Copyright Life Technologies®, Inc., 1999

FOCUS® is published triannually by Life Technologies, Inc.

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ABOUT THE COVER: Transfection in 96-well plates.(See page 58).

Essential Technologies for the Science of Life™

®

L IPOFECTAMINE 2000 Reagenttransfects cells at very high effi-ciencies, resulting in high levels ofrecombinant protein expression (1).

The protocol for transfection of cells withLIPOFECTAMINE 2000 Reagent involvesvery few steps and can be performed inserum-containing medium. Therefore,this new reagent is especially suited fortransfections in the 96-well format, whichis ideal for high-throughput protocols.

In this report, we optimized transfectionconditions for 96-well plates with the 3 celllines commonly used in high-throughputprotocols—CHO, COS, and 293—usingeither a "Standard Protocol," where cells areplated the day before transfection, or a"One-day Protocol," where a cell suspensionis added to the DNA-reagent complexesprepared in the wells. The sensitivity ofthese protocols to measure gene activity incDNA libraries is also shown.

METHODSCELL CULTURE. All cell lines, culture media,

sera, and reagents were from LifeTechnologies. CHO-S (Cat. No. 11619),COS-7L (Cat. No. 11622), and 293-H(Cat. No. 11631) cells (2) were cultured inDulbecco’s MEM (D-MEM High Glucose:4,500 mg/L D-glucose, with 584 mg/L L-glutamine and 15 mg/L phenol red) sup-plemented with 0.1 mM nonessentialamino acids (NEAA) and 10% FBS. Cellcultures were maintained at 37°C in ahumidified, 5% CO2 incubator. For COSand 293 cells, poly-D-Lysine coated plateswere used for transfection protocols.

TRANSFECTION. For the Standard Protocol,cells were plated in 96-well plates the daybefore transfection in 100 µl of growthmedium. Several seeding densities weretested for each cell type (CHO-S, 2 × 104

to 4 × 104; COS-7L, 1.5 × 104 to 2.5 × 104;293-H, 4 × 104 to 6 × 104 cells/well).

t r a n s f e c t i o n

58 F O C U S ( 1 9 9 9 ) V O L U M E 2 1 N U M B E R 3

Jean-Pierre PichetValentina Ciccarone

Molecular Biology Research and Development

Life Technologies, Inc.Rockville, Maryland 20849Transfection of Mammalian

Cells in 96-Well Plates withLIPOFECTAMINE ™2000 Reagent

FIGURE 1. Transfection of cells using the standard protocol. Cells were transfected with pCMV•SPORT-βgal DNA and LIPOFECTAMINE 2000Reagent. At 24 h after complex addition, duplicate plates were assayed with X-gal (panel A) and ONPG (panel B). Results from the optimalseeding density for each cell line are shown: 2 × 104 for CHO-S, 2.5 × 104 for COS-7L, and 5 × 104 for 293-H.

18,000

16,000

8,000

10,000

12,000

14,000

6,000

4,000

2,000

00.2 0.4 0.6 0.8 1.0 1.2

80160

240320

ng β

-gal

/cm

2

DNA(ng)

320 –

240 –

160 –

80 –

0.2 0.4 0.6 0.8 1.0 1.2

Panel A

293-H Cells

COS-7L Cells

CHO-S Cells

Panel B

320 –

240 –

160 –

80 –

0.2 0.4 0.6 0.8 1.0 1.2

320 –

240 –

160 –

80 –

0.2 0.4 0.6 0.8 1.0 1.2

3,000

2,500

500

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1,500

2,000

00.2 0.4 0.6 0.8 1.0 1.2

80160

240320

ng β

-gal

/cm

2

DNA(ng)

0.2 0.4 0.6 0.8 1.0 1.280

160240

320

ng β

-gal

/cm

2

DNA(ng)

4,500

4,000

2,000

2,500

3,000

3,500

1,500

1,000

500

0

DNA

(ng)

DNA

(ng)

DNA

(ng)

LIPOFECTAMINE 2000 Reagent (µl)






pCMV•SPORT-βgal (Cat. No. 19586)and pCMV•SPORT6-βgal-neo plasmidDNA were purified using the CONCERT™

High Purity Plasmid Purification MaxiprepSystem (Cat. No. 11452). For each well,DNA and LIPOFECTAMINE 2000Reagent (Cat. No. 11668) were first dilutedseparately in 25 µl OPTI-MEM® I ReducedSerum Medium without serum. Thediluted LIPOFECTAMINE 2000 Reagentwas incubated for 5 min at room tempera-ture, before mixing with the diluted DNA.The DNA-reagent complexes, in 96-wellplates, were incubated at room temperaturefor 20 min for complex formation. Usingduplicate plates for each cell line, 50 µl ofcomplex was added directly to the cells intheir growth medium and gently mixed.

For the rapid, One-day Protocol,DNA-reagent complexes were prepared asdescribed above in 96-well plates. Cells weretrypsinized, and a 100-µl cell suspensionwas added to the complexes in wells.

CDNA LIBRARY SCREEN. pCMV•SPORT6-βgal-neo was serially diluted into totalplasmid DNA from a human brain cDNAlibrary. The total DNA amount transfectedwas constant in all wells at 320 ng. Theamount of pCMV•SPORT6-βgal-neoDNA in the wells varied from 156 pg to320 ng. The transfection was performedwith the Standard Protocol.

β-GAL ACTIVITY. At 24 h post-transfection,the cells were rinsed with D-PBS and eitherfixed and stained in situ with X-gal (3) orlysed and harvested in 150 µl of 0.1 MTris-HCl (pH 8.0), 0.1% Triton®X 100to measure β-gal enzymatic activity usingONPG (4). Cells were frozen in lysis bufferat −80°C for at least 1 h prior to assay.

RESULTS AND DISCUSSIONSTANDARD PROTOCOL. To determine optimal

conditions for transfection in a 96-well plate,LIPOFECTAMINE 2000 Reagent and DNA

concentration were evaluated at 3 cell den-sities. Good transfection efficiencies wereobtained with the 3 cell lines, and optimalranges were identified (figure 1, table 1).

ONE-DAY PROTOCOL. A more rapid, One-dayProtocol where the complexes are preparedin a 96-well plate and a cell suspension isadded directly to the complexes was evalu-ated. In this protocol, it is not necessary toplate the cells the day before transfection.Ranges of DNA and reagent concentrationsat 3 cell densities were evaluated to deter-mine optimal conditions. The optimal cell

concentration for the rapid, One-dayProtocol was ∼2.5 times higher than for theStandard Protocol (figure 2, table 2). Themaximum activity for 293 and COS-7 cellswas lower than with the Standard Protocol(∼40% for 293 and ∼50% for COS-7).However, the expression levels were good,and time savings may provide an advantagein some applications. For CHO cells, theactivity was ∼70% lower, so this rapid pro-tocol may not be as useful with this cell line.

CDNA LIBRARY SCREENING. The standard pro-tocol was used to determine the sensitivity



TABLE 1. Optimal transfection conditions of cells in 96-well plates using the standard protocol.

TABLE 2. Optimal transfection conditions of cells with the rapid, one-day protocol.

FIGURE 2. Transfection of cells using the rapid, one-day protocol. DNA-LIPOFECTAMINE 2000 Reagent complexes were prepared in 96-wellplates and cell suspension added to each well. At optimal conditions, the cell concentrations were 1.2 × 105 cells/well for 293-H cells and 6 × 104

cells/well for COS-7L cells in a total volume of 100 µl of growth medium.

Cell Line Seeding Density DNA LIPOFECTAMINE 2000(cells/well) (ng/well) Reagent (µl/well)

CHO-S 5 × 104 320 0.8

COS-7L 6 × 104 320 0.6–0.8

293-H 1.2 × 105 320 0.4

Cell Line Seeding Density DNA LIPOFECTAMINE 2000(cells/well) (ng/well) Reagent (µl/well)

CHO-S 2 × 104 240 1

COS-7L 2.5 × 104 320 1

293-H 5 × 104 320 1

18,000

16,000

8,000

10,000

12,000

14,000

6,000

4,000

2,000

00.2 0.4 0.6 0.8 2.0 1.2

80160

240320

ng β

-gal

/cm

2

DNA(ng)

293-H Cells

3,000

2,500

500

1,000

1,500

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00.2 0.4 0.6 0.8 2.0 1.2

80160

240320

COS-7L Cells

ng β

-gal

/cm

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DNA(ng)

LIPOFECTAMINE 2000 Reagent (µl) LIPOFECTAMINE 2000 Reagent (µl)


of detection of gene expression in a cDNAlibrary. Expression was detected with smallamounts of β-gal plasmid (figure 3), sug-gesting that this protocol may be used forscreening to detect specific gene expressionin cDNA libraries. Detection levels willdepend on the vector used and the assaysensitivity. For example, the better COS-7Lsensitivity was due to the SV40 origin ofreplication in the plasmid allowing higherexpression levels in the SV40-transformedCOS-7L cells. Other detection methods (i.e.luminescence) may allow greater sensitivity.

In summary, LIPOFECTAMINE 2000Reagent is highly efficient for transfectionof cells in a 96-well format. Starting withthe protocols described here, transfectionconditions can be easily established forautomated or robotic systems in high-throughput screening applications.

ACKNOWLEDGEMENTSThe authors thank Dr. Ray Hadley and

Dr. Chris Gruber for the brain librarycDNA.

REFERENCES1. Ciccarone, V., Chu, Y., Schifferli, K., Pichet, J.-P.,

Hawley-Nelson, P., Evans, K., Roy, L., and Bennett, S.(1999) FOCUS 21, 54.

2. Roy, L., Cates, S., Schifferli, K., Pichet, J.P., Ciccarone,V., Bennett, S., and Hawley-Nelson, P. (1999) FOCUS 21,62.

3. Sanes, J.R., Rubenstein, L.R., and Nicolas, J.F. (1986)EMBO J. 5, 3133.

4. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, New York. SecondEdition, p. 16.66.

FOCUS


Transfection of Mammalian Cells continued

320 160 80 40 20 10 5 2.5 1.25 0.625 0.313 0.1560

6,000

5,000

4,000

3,000

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β-g

al/c

m2

Target DNA (ng)

320 160 80 40 20 10 5 2.5 1.25 0.625 0.131 0.1560

12,000

10,000

8,000

6,000

4,000

2,000

ng β

-gal

/cm

2

Target DNA (ng)

FIGURE 3. Detection of gene expression in a cDNA library. Cells plated at their optimal seeding density were transfected with serially diluted pCMV•SPORT6-βgal-neo added to plasmid DNA from a cDNA library. Thetotal amount of DNA transfected was always 320 ng. Wells in each column of the plate contained the same amount of pCMV•SPORT6-βgal-neo DNA. Duplicate plates were assayed with X-gal (panel A) and ONPG (panelB). Data in panel B are the mean ± SD for N = 8.

320 160 80 40 20 10 5 2.5 1.25 0.625 0.313 0.156

320 160 80 40 20 10 5 2.5 1.25 0.625 0.313 0.156

Panel A

COS-7L Cells

293-H Cells

Panel B

Target DNA (ng)

Target DNA (ng)



When starting transfections inyour lab, the general guidelinesto follow in selecting a reagentare summarized at the right.

The table below contains recommendedcationic lipid reagents for high-efficiencytransfection of key cell lines.* Please consultour web site (www.lifetech.com) for themost current additions to our recom-mended reagent table.

Cationic Lipid Reagent Selection

Application Recommended Reagent

Difficult-to-transfect cells LIPOFECTAMINE 2000 Reagent

Most adherent cells LIPOFECTAMINE 2000 Reagent or LIPOFECTAMINE PLUS Reagent

Adherent cells in the presence of serum LIPOFECTAMINE 2000 Reagent

Suspension cells DMRIE-C Reagent

Insect cells CELLFECTIN® Reagent

Endothelial cells LIPOFECTIN® Reagent

RNA DMRIE-C Reagent

Oligonucleotides LIPOFECTIN Reagent, LIPOFECTAMINE Reagent, or CELLFECTIN Reagent

Cell Line Mammalian Cell Type Recommended Reagent

293-F Human kidney LIPOFECTAMINE 2000 Reagent

293-H Human kidney LIPOFECTAMINE 2000 Reagent

BE(2)C Human neuroblastoma LIPOFECTAMINE 2000 Reagent (without serum)

BHK-21 Hamster kidney LIPOFECTAMINE PLUS Reagent (without serum)

CHO-K1 Hamster ovary LIPOFECTAMINE 2000 Reagent

CHO-S (adherent) Hamster ovary LIPOFECTAMINE 2000 Reagent

CHO-S (suspension) Hamster ovary DMRIE-C Reagent(In CD-CHO Medium) LIPOFECTAMINE 2000 Reagent

COS-1 Monkey kidney LIPOFECTAMINE 2000 Reagent (without serum)LIPOFECTAMINE PLUS Reagent

COS-7L Monkey kidney LIPOFECTAMINE 2000 Reagent

Fibroblasts Human primary passaged LIPOFECTAMINE 2000 Reagent (without serum)LIPOFECTAMINE PLUS Reagent (without serum)

HeLa Human cervical cancer LIPOFECTIN with PLUS Reagent

HT-29 Human colon cancer LIPOFECTAMINE 2000 Reagent (without serum)LIPOFECTAMINE PLUS Reagent (without serum)

HT-1080 Human fibrosarcoma LIPOFECTAMINE 2000 Reagent (with serum)

HUAEC (primary) Human endothelial LIPOFECTIN Reagent

HUVEC (primary) Human endothelial LIPOFECTIN Reagent

Keratinocytes (In Keratinocyte-SFM) Human primary passaged LIPOFECTAMINE PLUS Reagent

MDCK Dog kidney LIPOFECTAMINE 2000 Reagent

MRC-5 Human lung LIPOFECTAMINE 2000 Reagent (without serum)LIPOFECTAMINE PLUS Reagent (without serum)

NIH-3T3 Mouse fibroblasts LIPOFECTAMINE PLUS Reagent

PC-12 Rat pheochromocytoma LIPOFECTAMINE 2000 Reagent

SK-BR3 Human breast cancer LIPOFECTAMINE 2000 Reagent (with serum)LIPOFECTAMINE PLUS Reagent (without serum)

Vero Monkey kidney LIPOFECTAMINE 2000 Reagent (without serum)

Cell Line Insect Cell Type Recommended Reagent

D.Mel-2 Drosophila melanogaster CELLFECTIN Reagent

Sf9 Spodoptera frugiperda CELLFECTIN Reagent

Sf21 Spodoptera frugiperda CELLFECTIN Reagent

* Protocols for these transfections are in the TECH-ONLINE section of our web site at www.lifetech.com and available from the TECH-LINE.


Chinese Hamster Ovary (CHO) (1)cells, COS-7 (2) cells, and HEK 293(3) cells are among the most com-monly used mammalian cell lines

for transfection, expression, and large-scaleproduction of recombinant proteins. Whilethese cell lines have been available for manyyears, the commercial lines have not alwaysbeen easily transfected. Since these immor-talized cell lines are aneuploid, sublineshave evolved in various laboratories thatare subtly different from the cells originallyisolated, cultured, and banked at AmericanType Culture Collection (ATCC) or othercell banks. As a result, variability in trans-fection efficiency has been observed. Someresearchers have selected for more easilytransfected variants. Life Technologies hasused several better-transfecting sublines toisolate clones exhibiting high transfectionefficiency. In this paper, we show that thesecell lines exhibit higher transfection activitythan other available cells.

METHODSCELL CULTURE. All cell culture media, sera,

and reagents were from Life Technologiesunless otherwise noted. For transfection,cells were grown as adherent cultures inDulbecco’s MEM (D-MEM, high glucose:4,500 mg/L D-glucose, with 584 mg/LL-glutamine and 15 mg/L phenol red) sup-plemented with 0.1 mM non-essentialamino acids (NEAA) and 10% FBS at37°C in 5% CO2. ATCC cells were used<10 passages after thawing. We recommendLife Technologies cells for up to 30 passagesor 3 months post-thaw. After many passages,the transfection efficiency may change(9). Life Technologies cell lines used were293-H (Cat. No. 11631); 293-F (Cat. No.11625); CHO-S (Cat. No. 11619); andCOS-7L (Cat. No. 11622). The parentallines were obtained as follows: 293-H(from Leaf Huang at the University ofPittsburgh), 293-F (from Robert Horlick atPharmacopoeia), CHO-S (from RobertTobey at Los Alamos), and COS-7L (fromTom Livelli at Specialty Media).

TRANSFECTION. The day before the trans-fection, 1.5 × 105 CHO cells, 2.0 × 105

HEK 293 cells, and 8.0 × 104 COS-7 cellswere plated in 0.5 ml of growth medium ineach well of 24-well plates. Since 293 andCOS-7 cells are weakly adherent cells, theywere plated on poly-D-lysine (50 µg/well)coated plates. The cells were transfectedwith pCMV•SPORT-βgal DNA usingLIPOFECTAMINE™ 2000 Reagent (Cat.No. 11668) or LIPOFECTAMINE PLUS™

Reagent according to manufacturer’srecommendations. The DNA was preparedusing the CONCERT™ High PurityMaxiprep System.

For transfection with LIPOFECTAMINEPLUS Reagent (4), the DNA was precom-plexed with the PLUS Reagent by diluting0.8 µg DNA to 17 µl with D-MEM/

NEAA and adding 8 µl PLUS Reagent.The precomplexes were incubated at roomtemperature for 15 min. Meanwhile, 0 to5 µl of LIPOFECTAMINE Reagent werediluted to 25 µl with D-MEM/NEAA andincubated for 5 min at room temperature.The DNA/PLUS Reagent was mixed withthe diluted LIPOFECTAMINE Reagent andincubated for 15 min at room temperature.The growth medium was removed fromthe cells and replaced with 500 µl ofD-MEM/NEAA. The complexes wereadded to the cells and incubated for 4 h in37°C, 5% CO2. 500 µl of D-MEM/NEAAcontaining 20% FBS were added to the cells.

The transfections with LIPOFECTAMINE2000 Reagent (5) were performed in growthmedium (D-MEM/NEAA/10% FBS).0 to 6 µl of LIPOFECTAMINE 2000Reagent were diluted to 50 µl withDMEM/NEAA. DNA (0.8 µg) wasdiluted to 50 µl with D-MEM/NEAA.After 5 min, the diluted DNA was addedto the diluted LIPOFECTAMINE 2000Reagent and incubated for 15 min at roomtemperature. The complexes (100 µl) wereadded to each well, and the cells wereplaced at 37°C in 5% CO2.

b-GAL ACTIVITY. At 24 h after addition ofcomplexes, cells were rinsed once withDulbecco’s Phosphate Buffered SalineSolution and lysed in 0.1 M Tris-HCl (pH8.0), 0.1% Triton® X-100 (v/v) and frozenat −70°C overnight. Cell extracts werethawed and assayed for β-gal activity usingONPG (7). Protein was determined by aBradford assay. Alternatively, cells werefixed and stained in situ with X-gal (8).Stained cells were photographed using a10X objective on a Nikon inverted micro-scope with Hoffman optics. Percent stainedcells was determined by counting 3 fieldsand averaging.


Linda Roy, Sharon Cates,Kevin Schifferli, Jean-Pierre Pichet,

Valentina Ciccarone, Shelley Bennett,and Pamela Hawley-Nelson

Research and DevelopmentLife Technologies, Inc.

Rockville, Maryland 20849High Transfection Efficiency ofCloned Cell Lines

FIGURE 1. Expression of β-gal. 293 cells from Life Technologies(panel A) or ATCC (panel B) were transfected using LIPOFECTAMINE2000 Reagent and assayed after 24 h. The cells photographed arethe peak activity from a dose response.

A

B

RESULTS AND DISCUSSIONCell lines were evaluated for transfection

efficiency using 2 reagents. Results indicatedthat the clones selected for transfectabilityhad high peak transfection efficiency (figure1, table 1). The magnitude of differencefrom cells not selected for transfectionability was affected by the transfectionreagent, with LIPOFECTAMINE 2000Reagent demonstrating >95% trans-fected cells for the Life Technologies cells.This high efficiency was possible withtransfection in the presence of serum withLIPOFECTAMINE 2000 Reagent.

Similarly, the peak β-gal activity washigher in the clones selected for transfec-tion (figure 2). In contrast to the percent of

cells transfected, the CHO-S clone demon-strated almost twice the β-gal activity of theCHO-K1 cell line.

In summary, the CHO-S, 293-H, 293-F,and COS-7L cells were transfected betterthan ATCC cell lines. For added flexibility,these cells are provided adapted to serum-free medium and suspension culture. Thisfacilitates use of these cells for large-scaleprotein production after selection of a stablytransfected clone. For CHO-S cells, it alsoallows transient transfection in suspensionculture and the chemically defined CD-CHO Medium (6).

REFERENCES1. Puck, T. (1958) J. Exp. Med. 108, 945.

2. Glutzman, Y. (1981) Cell 23, 175.

3. Graham, F.L. (1977) J. Gen. Virol. 36, 319.

4. Shih, P.-J., Evans K., Schifferli, K., Ciccarone, V., Lichaa,F., Masoud, M., Lan, J., and Hawley-Nelson, P. (1997)Focus 19, 52.

5. Ciccarone, V., Chu, Y., Schifferli, K., Pichet, J.-P.,Hawley-Nelson, P., Evans, K., Roy, L., and Bennett, S.(1999) Focus 21, 54.

6. Schifferli, K., Jessee, J., and Ciccarone, V. (1999) Focus21, 16.

7. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, New York. SecondEdition, p. 16.66.

8. Sanes, J.R., Rubenstein, L.R., and Nicholas, J.F. (1986)EMBO J. 5, 3133.

9. Hawley-Nelson, P. and Shih, P.-J. (1995) Focus 17, 60.

FOCUS



Transfection Efficiency (% stained cells)LIPOFECTAMINE PLUS LIPOFECTAMINE 2000

Cell Line Source Reagent Reagent

COS-7 ATCC 35 ± 2 73 ± 11

COS-7L Life Technologies 74 ± 6 99 ± 0

293 ATCC 39 ± 9 73 ± 8

293-F Life Technologies 99 ± 0 99 ± 0

293-H Life Technologies 99 ± 0 99 ± 0

CHO-K1 ATCC 45 ± 3 86 ± 2

CHO-S Life Technologies 97 ± 2 96 ± 3

TABLE 1. Transfection efficiency. Cells were stained for β-gal at 24 h after addition of complexes.

FIGURE 2. Comparison of β-gal activity. Peak transfection activity of Life Technologies (■) or ATCC (□) cell lines was determined with ONPG24 h after addition of DNA/LIPOFECTAMINE 2000 Reagent complexes.

160

140

120

100

80

60

40

20

0293 ATCC293-H293-F

HEK 293

45

40

35

30

25

20

15

10

5

0COSCHO

The Help Box from YourTechnical Support & Training Team

Q. How does LIPOFECTAMINE 2000 Reagentimprove transfection compared to otherreagents?

A. Higher levels of protein expression have beenobserved in most of the cell lines tested. Inaddition, the streamlined protocol allows theaddition of complexes directly to cells withoutchanging media.

Q. Why do you recommend using mediumwithout antibiotics during transfection?

A. While penicillin and streptomycin are nottoxic to eukaryotic cells, the increased cell per-meability occurring during transfection leadsto much higher levels of antibiotics gettinginto cells. This can lead to higher cell death.

Q. Can I use the same amount of any cationiclipid reagent for my cell line?

A. No. Optimize the amount of each reagent.

Q. What is the shelf life of cationic lipid reagents?A. Stored at 4°C in a closed container, they are

stable for 12 months. Do not freeze the reagents.

Q. Is it necessary to use medium without serumduring transfection?

A. No. It is essential to form the DNA/cationiclipid reagent complex in the absence ofserum, because proteins can interfere withcomplex formation. Once the complexes areformed, they can be added to cells in serum-containing medium.

ng β

-Gal

/µg

prot

ein

ng β

-Gal

/µg

prot

ein

Fluorescence-based DNA electro-phoresis systems are used often ingenotyping applications to analyzepolymorphic markers whose alleles

differ by discrete size increments. To deter-mine the sizes of the unknown fragments,the instruments and software usuallyrequire that the size standard be elec-trophoresed in the same lane or capillarywith each sample.

For gel-based systems, sharkstooth combsare used for higher sample throughput andeasier loading of the thin gels. For the soft-ware to distinguish adjacent lanes from oneanother, it becomes necessary to load everyother lane of the gel, electrophorese thesamples briefly, and load the remaininglanes of the gel. If all lanes of the gel wereloaded at the same time, the DNA bands ofthe sizing ladder would appear as continuouslines across the entire gel and adjacent laneswould not be distinguishable.

An alternative to time-consuming mul-tiple gel loadings is to use DNA ladderswith different band sizes in adjacent lanes.This paper describes fluorescently labeledDNA ladders that can be used individually oras a dual-ladder configuration for simplifiedloading of gels with sharkstooth combs.

METHODSFor gel electrophoresis, the GENOTYPE

TAMRA 50-500 DNA Ladder (Cat. No.11726) and the GENOTYPE TAMRA60-500 DNA Ladder (Cat. No. 11727)were analyzed on the ABI 377XL

Automated Sequencer and were used todetermine the size of PCR products ampli-fied from dinucleotide repeat microsatellitemarkers using primer sets from Genethonpublished sequences as previously described(1). For analysis, 1.5 µl of sample or waterwere mixed with 2.75 µl of deionizedformamide, 0.25 µl of DNA ladder, and0.5 µl of blue dextran solution (50 mg/mlblue dextran, 10 mM EDTA). Themixtures were heat denatured at 90°Cfor 2 min, cooled on ice, and 1.5 µl wereelectrophoresed on a 36-cm, 4.25%(GEL-MIX® 4.25FA, Cat. No. 10773) poly-acrylamide sequencing gel using a 36-lanesharkstooth comb. Data were analyzedusing GeneScan® 3.1.1 software.

For capillary electrophoresis, the lad-ders were analyzed on a POP-6 matrixunder denaturing conditions using an ABI310 Capillary Genetic Analyzer. 0.25 µl ofthe DNA ladder was mixed with 12 µl ofdeionized formamide, heat denatured at95°C for 5 min, cooled on ice, and theentire sample injected. Data were analyzedusing GeneScan 3.1.1 software.

e l e c t r o p h o r e s i s


Heather Jordan and Joseph SolusResearch and Development

Life Technologies, Inc.Rockville, Maryland 20849DNA Ladders for Fluorescent

Fragment Analysis

Data

Poi

nt

1,000

1,500

2,000

2,500

3,000

3,500

4,000

4,500

5,000

50050 100 150 200 250 300 350 400 450

Size (bp)

R2 = 0.9994

FIGURE 2. Linear correlation of the GENOTYPE TAMRA 50-500 DNA Ladder. The GENOTYPE TAMRA 50-500 DNA Ladder was analyzed byelectrophoresis on an ABI 377 gel. The fluorescent fragments were detected as they passed by the scanning laser during electrophoresis. The"Data Point" (Y axis) is a measure of the number of scans across the gel by the laser prior to detection of the fragment and thus corresponds tothe migration rate of the fragment.

FIGURE 1. The GENOTYPE DNA Ladders. Alternating lanes of a4.25% denaturing polyacrylamide gel were loaded with theGENOTYPE TAMRA 60-500 DNA Ladder and the GENOTYPETAMRA 50-500 DNA Ladder.

b –500 –490 –480 –

460 –

440 –

420 –

400 –

380 –

360 –350 –340 –

320 –

300 –

280 –

260 –

240 –

220 –

200 –

180 –

160 –

140 –

120 –

100 –90 –80 –

60 –

– b – 500 – 490

– 470

– 450

– 430

– 410

– 390

– 370

– 350

– 330

– 310– 300

– 290

– 270

– 250

– 230

– 210– 200– 190

– 170

– 150

– 130

– 110

– 90

– 70

– 50

RESULTS AND DISCUSSIONThe GENOTYPE TAMRA DNA

Ladders were analyzed in the dual-ladderconfiguration (figure 1). All lanes wereloaded simultaneously and the 2 distinctband patterns were visible, allowing auto-mated, error-free lane tracking by the soft-ware. The migration of bands was linearwith respect to fragment size (figure 2).

Several dinucleotide repeat microsatel-lite markers amplified from human DNAas previously described (1) were sized withthe GENOTYPE DNA Ladders. Themeasured sizes calculated using theGENOTYPE DNA Ladders (table 1) wereconsistent with data obtained previouslyusing commercially available ladders (datanot shown).

The GENOTYPE TAMRA DNALadders were analyzed by capillary elec-trophoresis (figure 3). The electrophero-grams illustrate the wide size range andeven peak intensities of the ladders.Orientation markers (doublets and triplets)of the GENOTYPE DNA Ladders areindicated by arrows.

In summary, fluorescent GENOTYPEDNA Ladders provide a dual-ladder formatto facilitate automated lane tracking andeliminate multiple gel loadings. The laddersaccurately sized human DNA markersusing gel-based or capillary-based (data notshown) electrophoresis systems.

EDITOR’S NOTE: The ladders are also avail-able labeled with ROX (GENOTYPE ROX50-500 DNA Ladder, Cat. No. 11728, andGENOTYPE ROX 60-500 DNA Ladder,Cat. No. 11729).

REFERENCE1. Jordan, H., Darfler, M., and Solus, J. (1999) FOCUS 21, 1.

FOCUS


e l e c t r o p h o r e s i s

TABLE 1. Size determination of dinucleotide repeat microsatellite markers. Markers were amplified using Genethon published sequences,and the sizes of the amplicons were determined using the GENOTYPE TAMRA DNA Ladders on an ABI 377XL DNA Sequencer. The expectedsize range was determined by Genethon from analysis of a large number of alleles for each locus, but may not account for all alleles in thehuman population.

Calculated Size (bp)Expected GENOTYPE TAMRA GENOTYPE TAMRA

Locus Size Range (bp) 50-500 DNA Ladder 60-500 DNA Ladder

D1S196 allele 1 267–297 272.05 272.12allele 2 273.13 273.13

D1S220 allele 1 231–251 245.35 245.46allele 2 247.31 247.46

D1S234 allele 1 226–238 230.94 230.99allele 2 232.91 232.88

D1S235 allele 1 175–195 173.90 173.77allele 2 174.82 174.79

D9S167 allele 1 260–286 264.72 264.90allele 2 272.70 272.70

D16S671 allele 1 338–372 369.08 369.72

FIGURE 3. Capillary electrophoresis of GENOTYPE TAMRA DNA Ladders.

1,200 –-

800 –-

400 –-

0 –

1,200 –-

800 –-

400 –-

0 –

GENOTYPE TAMRA 50–500 DNA Ladder

GENOTYPE TAMRA 60–500 DNA Ladder

Fluo

resc

ence

Inte

nsity

– – – – – – – – – –

50 100 150 200 250 300 350 400 450 500

Size (b)

p c r


ABSTRACTThe macrophage is a central cell in inflam-

mation, host defense, and wound healing.Macrophages are active as phagocytes andproduce mediators that attract other leuko-cytes and/or initiate reconstruction of tissuelesions. We describe methods to study mediatorproduction from the murine macrophage cellline RAW 264.7, with a special focus onchemokines. Applying an easy-to-use one-stepRT-PCR procedure, we demonstrate the rapidinduction of a variety of cytokine/chemokinemRNAs by LPS in the macrophages. In mostcases, the message data correlated with ELISAresults on the release of the respective mediators.

The macrophage plays an importantrole in immune responses (1). It ishighly versatile, very mobile, andresponsive to all types of inflamma-

tory stimuli. Particularly, monocytes andmacrophages are able to release mediators(e.g., cytokines) that activate and modulateother immune and non-immune cells.Studying cytokine release from macrophagesmay help us understand fundamentalmechanisms of inflammation. Notably, themacrophage is central not only in hostdefense but also in wound healing (2).ELISA is used to measure cytokine induc-tion. However, ELISAs require specificantibodies, which are not always availablefor newly discovered cytokines. Anothermethod to study the induction of a mediatoris RT-PCR. Furthermore, mRNA data giveadditional information when used withELISA, e.g., when only transcription of amediator is induced but not translation.

We applied one-step RT-PCR as well asELISA in a simple in vitro system of macro-phage cytokine/chemokine induction. Thesystem is the widely used murine cell lineRAW 264.7 stimulated with the classicalbacteria-derived stimulus lipopolysaccha-ride (LPS). We show that the 2 methods

produce corresponding and complemen-tary results. Importantly, one-step RT-PCRwas easy to use and unfailing.

METHODSSTIMULATION OF CELLS. Murine RAW 264.7

macrophages (ATCC) were grown inDulbecco’s Modified Eagle Medium (D-MEM) with 10% heat-inactivated FBS andpenicillin/streptomycin at 37°C, 5% CO2

in a humidified atmosphere. Cells wereroutinely passaged by scraping with a splitratio of 1:5.

For ELISA assays, RAW cells wereseeded at 2 × 105 cells/ml in 200 µl ofmedium/well on flat-bottomed 96-wellmicrotiter plates in the afternoon the daybefore stimulation. The next morning,cells were stimulated with 1 µg/ml LPS(Salmonella abortus equi) by adding the LPS

in 20 µl medium/well. The same volume ofmedium without LPS was added to controlwells. After 24 h, supernatants were har-vested and stored at –20°C, since previousexperience had shown that most mediatorsreached saturation at 24 h. Also, rapidlyproduced cytokines (e.g., TNF-α) accumu-lated stably in the culture medium.

For RT-PCR analysis, stimulation wasas described above, but in 1 ml/well on24-well plates. Based on previous expe-rience, cells were harvested 5 h after stimu-lation, at a time when macrophages initiatethe liberation of most of the mediatorsmeasured. Supernatants were discarded and170 µl TRIZOL® Reagent were added perwell. The cell lysates were harvested afterscratching the well with a 200-µl pipettetip and multiple resuspensions. Lysates of6 wells were pooled in a 1.5-ml tube. Total

One-Step RT-PCR to DetectCytokine/Chemokine Induction inMacrophages

Monique Bongers, Ekke Liehl,and Johannes Barsig

Novartis Research InstituteBrunner Str. 59

1235 Vienna, Austriae-mail: [email protected]

Primer (Reference) Sequence Product Size (bp) Tm (°C)

β-actin-s ATG GGT CAG AAG GAT TCC TAT GTG 359 56.0β-actin-as CTT CAT GAG GTA GTC AGT CAG GTC 57.0

IP-10-s (3) CGC ACC TCC ACA TAG CTT ACA G 431 54.1IP-10-as (3) CCT ATC CTG CCC ACG TGT TGA G 58.1

KC-s (3) GAC GAG ACC AGG AGA AAC AGG G 530 56.5KC-as (3) AAC GGA GAA AGA AGA CAG ACT GCT 54.7

MIP-2-s (3) TGG GTG GGA TGT AGC TAG TTC C 466 54.6MIP-2-as (3) AGT TTG CCT TGA CCC TGA AGC C 58.3

MCP-1-s (3) GGA AAA ATG GAT CCA CAC CTT GC 582 58.3MCP-1-as (3) TCT CTT CCT CCA CCA CCA TGC AG 59.9

MIP-1α-s (3) GAA GAG TCC CTC GAT GTG GCT A 561 54.9MIP-1α-as (3) CCC TTT TCT GTT CTG CTG ACA AG 54.8

MIP-1β-s (3) CCA CAA TAG CAG AGA AAC AGC AAT 540 54.4MIP-1β-as (3) AAC CCC GAG CAA CAC CAT GAA G 59.7

TNF-α-s (4) TCT CAT CAG TTC TAT GGC CC 231 49.0TNF-α-as (4) GGG AGT AGA CAA GGT ACA AC 42.5

IL-10-s (5) CTG GAC AAC ATA CTG CTA ACC GAC 300 54.6IL-10-as (5) ATT CAT TCA TGG CCT TGT AGA CAC C 57.2

IL-6-s (4) GTT CTC TGG GAA ATC GTG GA 227 51.4IL-6-as (4) TGT ACT CCA GGT AGC TAT GG 44.3

IL-1β-s (4) TTG ACG GAC CCC AAA AGA TG 223 54.7IL-1β-as (4) AGA AGG TGC TCA TGT CCT CA 49.3

GM-CSF-s (6) ATG TGG CTG CAG AAT TTA CTT TTC CT 435 57.7GM-CSF-as (6) TGG GCT TCC TCA TTT TTG GCC TGG T 66.0

TABLE 1. RT-PCR primers. The β-actin primers were designed by the author. According to the references, primer sets were designed to amplifya DNA fragment from at least 2 exons of each target so that the fully spliced, mature mRNA product could be distinguished from the unsplicedmRNA or contaminating DNA products. “s” is the sense primer and “as” is the antisense primer.


p c r

RNA was isolated as recommended by themanufacturer. RNA was dissolved in 22 µlof nuclease-free water and stored at −70°C.The quantity of RNA was determined byA260.

ONE-STEP RT-PCR. RT-PCR was performedusing the SUPERSCRIPT™ ONE-STEPRT-PCR System. Briefly, one-step RT-PCRwas performed on 1 µg RNA using 50pmol each primer (table 1) in a 50-µlreaction. Incubation was at 60°C for 30min; 94°C for 1 min; then 35 cycles of94°C for 15 s, 60°C for 30 s, and 72°C for60 s. A final extension at 60°C for 7 min wasperformed. After 20, 25, 30, and 35 cycles,30-s pauses were programmed to obtain5 µl of sample. 2 µl loading buffer and 13 µlwater were added to samples, and 10 µlwere separated on a 2% agarose gel in 1XTBE with 0.5 mg/L ethidium bromide at110 V. Bands were visualized with a GIBCOBRL® UV Transilluminator, and imagesand densitometry analysis used the Kodak®

Digital Science EDAS 120 System. Finalimages were composed using the Corel 7software package.

ELISA. Antibodies and recombinantcytokines were used at the concentrationsrecommended by the suppliers. A standardELISA was performed with NUNCMaxisorp F96 Immunoplates, Certifiedusing an overnight coating, 1 h blocking(10% FBS and 0.05% Tween 20® in PBS;then 2 wash steps with 0.05% Tween 20 inPBS), 4 h sample incubation (then 4 washsteps), 1 h biotinylated antibody (then 4wash steps), 30 min Streptavidin-PODconjugate (then 5 wash steps), and 10 to30 min BM Blue incubation. Each platecontained a standard curve (10 ng/ml to10 pg/ml in duplicate) and 2 blank wellswith blocking buffer. The plates were readwith a SPECTRAmax Reader, and the datawere analyzed using the Softmax Pro 2.2.1.Detection limits for the ELISAs were 10 to

50 pg/ml, and blank values were belowOD 0.1. For samples, ODs between 0.1and 2.5 were used by analyzing samples inthe appropriate dilutions.

RESULTS AND DISCUSSIONCYTOKINE/CHEMOKINE MRNA INDUCTION BY LPS.

One-step RT-PCR was used to investigatemRNA induction by LPS. First, as acontrol, β-actin products were analyzed. Asexpected, the level of β-actin products wassimilar with and without LPS at all cyclenumbers (figure 1). The amount of β-actinproduct increased with increasing cyclenumber and was saturated by 30 cycles.These data indicate that comparableamounts of RNA were subjected toRT-PCR from control and LPS-incubatedcells. The importance of sampling atvarious cycle numbers was more evident

with induction of MIP-1α (macrophageinflammatory protein-1α). 20 cyclesshowed induction of message in LPS-stimulated cells, but at 25 cycles theunstimulated cells also had a signal, whichagrees with the ELISA data below. By30 cycles, saturation was reached forLPS-stimulated cells, and the differencebetween control and LPS stimulation wasnearly gone. Based on these results (andin accordance with the literature), thismethod is close to semi-quantitative, aslong as cycle numbers are controlled.Furthermore, sampling from one tubeduring RT-PCR kept RNA consumptionand costs low.

The ONE-STEP RT-PCR System canbe used for semi-quantitative and evenquantitative RT-PCR by adding internalcontrols and adjusting target signals to those,

FIGURE 1. mRNA induction in RAW 264.7 cells analyzed by one-step RT-PCR. Samples were taken from each tube after 20, 25, 30, and35 cycles for β-actin and MIP-1α. Lane 1. Cells not treated with LPS. Lane 2. Cells treated with 1 µg/ml LPS. The lanes at 35C are controls donewithout addition of RT. Lane C. Control without RNA. The graphs are densitometry of the gels.

Sign

al In

tens

ity [%

max

]

100

80

60

40

20

0

30 352520

Cycle Number

Sign

al In

tens

ity [%

max

]

100

80

60

40

20

0

30 352520

Cycle Number

–LPS

+LPS

20 25 30 35 35C 20 25 30 35 35C

600 bp –

Control1 2 1 2 1 2 1 2 C 1 2

Control1 2 1 2 1 2 1 2 C 1 210

0 bp

DNA

Lad

der

β-actin MIP-1α

p c r


or by using kinetic methods to producequantitative results. However, these methodsare more demanding in terms of equipmentand experience of personnel compared tothe procedures described here, which mayhence be more suitable to start with.

mRNA induction after LPS stimulationwas investigated for selected cytokines/chemokines. These mediators were inducedby the treatment, except for KC (neu-trophil-specific chemokine homologousto human GRO-α) (figure 2). All PCRproducts had the predicted sizes. Most ofthe cytokine messages were constitutivelyexpressed at low levels in unstimulated cellsand were highly upregulated by LPS. Theseexperiments show that one-step RT-PCR ishighly sensitive, the chosen primers workedunder these conditions, and RAW 264.7macrophages are useful to study inductionof a variety of cytokines/ chemokines.

CYTOKINE/CHEMOKINE RELEASE AFTER STIMULATION

WITH LPS. Induction of mRNA does notalways mean that the protein is producedand released. Mediator release from LPS-stimulated macrophages was examined byELISA.

LPS induced the production of most ofthe mediators analyzed (table 2), with someinteresting exceptions. IL-10 was notupregulated by LPS, even though low butsignificant mRNA was seen (see figure 2;note the high cycle number). Possibly,IL-10 released at a low level immediatelybound in an autocrine manner to receptorson the cells, thus prohibiting detection byELISA of supernatants. Also, IL-1β was notreleased from the RAW 264.7 cells (not inthe table), despite clear appearance ofmRNA. This can be explained by the com-plex mechanisms of IL-1β release from cells(7). The nascent protein lacks a signalsequence and is produced as a precursorthat has to be processed by IL-1 convertingenzymes. These enzymes have to be activated

themselves, possibly by a second signal. KCwas not produced by the macrophages, asseen with RT-PCR. However, induction ofKC mRNA and protein was seen in LPS-stimulated murine bone marrow-derivedmacrophages (data not shown).

In summary, RAW 264.7 cells are use-ful to study in vitro cytokine/chemokinerelease from murine macrophages (exceptfor KC). They can be obtained at highnumber and purity, and the cells are ratherquiescent when not stimulated, unlike thefrequently used peritoneal macrophages.Also, the SUPERSCRIPT ONE-STEPRT-PCR System provides a way for thenon-molecular biologist to easily andrapidly obtain mRNA induction data. Inmost cases, RT-PCR results of mRNAinduction corresponded to the ELISAresults on protein in the supernatant. Ourprotocol suggestions may speed up thework of other investigators interested inthis area.

REFERENCES1. Gordon, S. (1998) Res. Immunol. 149, 685.

2. DiPietro, L.A. (1995) Shock 4, 233.

3. Su, Y.-H., Yan, X.-T., Oakes, J., and Lausch, R. (1996)J. Virol. 70, 1277.

4. Zhou, H.-R., Yan, D., and Pestka, J.J. (1997) Toxicol.Appl. Pharmacol. 144, 294.

5. Colle, J.-H., Falanga, P.B., Singer, M., Hevin, B., andMilon, G. (1997) J. Immunol. Meth. 210, 175.

6. Ehlers, S., Mielke, M.E., Blankenstein, T., and Hahn, H.(1992) J. Immunol. 149, 3016.

7. Dinarello, C.A. (1998) Int. Rev. Immunol. 16, 457.

FOCUS

FIGURE 2. mRNA induction in RAW 264.7 cells analyzed by one-step RT-PCR. Samples were taken from each tube after 20, 25, 30, and 35cycles. Products are shown from cycle numbers where reactions behaved linearly (cycle numbers below mediator names). Lane 1. Cells nottreated with LPS. Lane 2. Cells treated with 1 µg/ml LPS. Controls without RNA or without RT were performed in each case and did not yieldproducts (data not shown).

600 bp –

1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

MIP-2 KC MCP-1 IP-10 MIP-1β IL-10 TNF-α IL-6 IL-1β GM-CSF25 35 25 25 25 35 20 35 35 35

100

bp D

NA L

adde

r

TNF-α IL-6 IL-10 MCP-1 MIP-1α

Control 0.01 ± 0.01 nd 0.09 ± 0.02 0.8 ± 0.4 7.6 ± 0.2

LPS-Stimulated 9.0 ± 1.7 82 ± 2 0.07 ± 0.01 55.5 ± 4.6 103 ± 33

MIP-1β MIP-2 KC IP-10 GM-CSF

Control 22 ± 2 1.2 ± 0.4 0.05 ± 0.01 1.7 ± 0.9 nd

LPS-Stimulated 183 ± 39 13 ± 9 0.03 ± 0.01 60 ± 8 0.88 ± 0.04

TABLE 2. Cytokine/chemokine production in RAW 264.7 cells. Data are mean ± S.D. in ng/ml for N = 3; nd = not detectable.

One-Step RT-PCR continued

ow are your oligonucleotidesmade?Oligonucleotides are made usingcomputer-controlled DNA syn-

thesizer and the patented parallel arraysynthesis method. The first nucleotide isattached to a solid support to anchor thegrowing DNA chain in the reaction col-umn. DNA synthesis consists of a series ofstandard β-cyanoethyl chemical reactions.

Each cycle consists of:• DEBLOCKING—The first nucleotide, attached

to the solid support via a chemical linkerarm, is deprotected by removing thetrityl-protecting group. This produces afree 5´-hydroxyl group to react with thenext nucleotide.

• COUPLING—The next nucleotide is added tothe reaction and couples (covalentlyattaches) to the first nucleotide.

• CAPPING—Any of the first nucleotide thatfailed to react is capped so that it will notplay a part in the subsequent synthesiscycles.

• OXIDATION—The bond between the firstnucleotide and the successfully coupledsecond nucleotide is oxidized to stabilizethe growing chain.

• DEBLOCKING—The 5´-trityl group is removedfrom the second nucleotide to prepare itfor further cycles.

Each cycle results in the addition of asingle nucleotide. A chain of nucleotides isbuilt by repeating the synthesis cycle untilthe desired length is achieved.

What is coupling efficiency? Coupling efficiency measures how

efficiently the DNA synthesizer added newnucleotides to the growing DNA chain. Ifevery available nucleotide on the DNAchain reacted successfully with the newnucleotide, the coupling efficiency wouldbe 100%. No chemical reaction is 100%efficient. Coupling efficiency for DNA

synthesis typically can reach ∼99%. Thismeans that at every coupling step approxi-mately 1% of the available nucleotides failto react.

Why is coupling efficiency important? It is used to determine the amount of

full-length oligonucleotide produced.

How do I determine the percentage offull-length oligonucleotide?

The percentage of full-length oligonu-cleotide depends on the coupling efficiency ofthe chemical synthesis. The average efficiencyis close to 99%.

To calculate the percentage of full-lengtholigonucleotide, use the formula:

Therefore, ∼79% of the oligonucleotide mole-cules in the tube are 25 bases long; the restare <25 bases.

If you are concerned about startingwith a preparation of oligonucleotide that isfull-length you may want to consider car-tridge, PAGE, or HPLC purification.

Is a small difference in coupling efficiencyimportant?

Since coupling efficiency is cumulativeduring DNA synthesis, even a smalldifference greatly affects the amount offull-length product. Table 1 shows the effectof a 1% difference in coupling efficiencyand how this influences the amount offull-length product for different-lengtholigonucleotides.

Why is trityl group analysis important toperform?

Every nucleotide added during DNAsynthesis has a dimethoxy trityl (trityl) pro-tecting group attached. This trityl groupprotects the nucleotide from undergoingunwanted chemical reactions during thesynthesis cycle and is removed immediatelybefore a new nucleotide is added. We usecontinuous trityl analysis to measurecoupling efficiency throughout the synthesis.

How do you measure coupling efficiency?The trityl group is colorless when

attached to a DNA base but gives a charac-teristic orange color once removed. Theintensity of this color is measured by spec-trophotometry and is directly related tothe number of trityl molecules present. Bycomparing the absorbance of tritylthroughout synthesis, it is possible tocalculate the percentage of nucleotidescoupling successfully and, hence, thecoupling efficiency.

Why do oligonucleotides sometimesrequire purification?

Depending on the application andoligonucleotide length, it may be necessaryto remove the oligonucleotide that is notfull length. Table 2 gives guidelines forthe minimum purity for a range ofapplications.


p c r : h e l p f u l t i p s

Helpful Tips for Custom Primers

Technical Services Part of the Technical Support and Training TeamLife Technologies, Inc.Rockville, Maryland 20849

H

% full length= (coupling efficiency)n-1,where n = total number of nucleotides.For example, the theoretical yield for a 25-merwould be:0.9924 = 0.79

Oligonucleotide Percent Full LengthLength

99% Coupling 98% Coupling

2 99 98

3 98.01 96.04

4 97.03 94.12

10 91.35 83.37

20 82.61 68.12

30 74.71 55.66

50 61.11 37.16

95 38.87 14.98

TABLE 1. Coupling efficiency affects the amount of full-lengthprimer.

When I order the 50-nmole scale, whydon’t I receive 50 nmoles of product?

Synthesis scales refer to the amount ofstarting material present, not the amountof product produced. This is the same forall manufacturers of synthetic DNA. Whena 50-nmole scale synthesis is specified,approximately 50 nmoles of the firstnucleotide are added to the DNA synthe-sizer. Losses occur during processing,transfer of material, and quality control.Oligonucleotide length, sequence, GCcontent, and coupling efficiencies can varythe yield of product. Purification of theoligonucleotide also affects final productyield as determined by final OD.

On average, the 50-nmole scale willsupply enough oligonucleotide for 1,000 to5,000 PCRs (for 100-µl reactions usingprimer concentration of 0.1 to 0.5 µM).

Primer length mattersThe length of the primer affects the

yield, as seen in table 3. The table assumes98% coupling efficiency, 100% recovery, and50% GC content using the 50-nmole scale.

How do I reconstitute my oligonucleotide?Dissolve the oligonucleotide in TE [10

mM Tris-HCl (pH 8.0), 1 mM EDTA].TE is recommended over deionized watersince the pH of the water is often slightlyacidic and can cause hydrolysis of theoligonucleotide.

To calculate the concentration of an oligonucleotide dissolved in 1 ml:

Concentration = A260 × Dilution Factor × Extinction Coefficient × Conversion Factors

Take the A260 reading by diluting 10 µl of the oligonucleotide with 990 µl of water (1:100 dilution).The A260 value is 0.14 OD. This oligonucleotide has an extinction coefficient of 4.9 nmole/OD.

Concentration = 0.14 OD/ml × 100 × 4.9 nmole/OD × 1 µmol/103 nmole × 103 ml/L= 69 µM

To calculate volume to dissolve an oligonucleotide in for a 100-µM solution:

Volume = Number of nmoles × (1 µmol/103 nmole)Desired Concentration × Conversion Factor (for L to µl)

For 24 nmole of an oligonucleotide:

Volume = [24 × (1/103)] × 106 µl100 µmol/L L= 240 µl

Helpful Tips for Custom Primers continued



Primer Size Expected OD Expected nmole(nucleotides)

10–20 4–7 40–32

20–30 7-9 32–27

30–40 9–10 27–22

40–50 10 22–18

50–60 10 18–15

Application Minimum Suggested Purity

PCR Standard

PCR using primers with critical 5´ sequences Cartridge(e.g., restriction endonuclease sites, RNA polymerase promoters)

First-strand cDNA synthesis for RT-PCR Standard

Fluorescent sequencing Standard

Cycle sequencing Standard

Isothermal sequencing Desalted

Site-directed mutagenesis Cartridge

First-strand cDNA synthesis for generation of libraries Cartridge

GENETRAPPER® screening PAGE

Production of cloning adapters Cartridge

Gel shift assays Cartridge

AFLP™ analysis Standard

CFLP™ analysis Desalted

Antisense HPLC purification is cited most frequently

TABLE 2. Suggested primer purity.

TABLE 3. Primer length affects yield.

Calculating primer concentration

How long can I store the oligonucleotide? The lyophilized oligonucleotide is sta-

ble at –20°C for at least 1 year. Theoligonucleotide dissolved in TE is stable forat least 6 months at –20°C or 4°C. Theoligonucleotide dissolved in water is stablefor at least 6 months at –20°C in theabsence of nucleases. Be sure the water usedis at neutral pH to avoid depurination. Donot store oligonucleotides in water at 4°C.

Analysis of oligonucleotides by electro-phoresis

Unlike large DNA molecules, sequencecan affect the migration rate of oligonu-cleotides. Oligonucleotides of the same sizecan migrate differently in denaturing poly-acrylamide gels, and these differences arecomposition and sequence dependent. • Since migration is sequence dependent,

comparison of an oligonucleotide to amolecular size standard or anotheroligonucleotide of known size is not areliable way to determine its size.

• Addition of 40% formamide (in additionto 8 M urea) to the acrylamide gel solu-tion removes most sequence dependentmigration effects.

[Ausubel, F.M., Brent, R., Kingston, R.E.Moore, D.D., Seidman, J.G., Smith, J. A.,and Struhl, K. (1994) Current Protocols inMolecular Biology, John Wiley and Sons,Inc., New York, NewYork.]

Use of ethidium bromide for detectionand quantitation of an oligonucleotide isnot reliable. The ability of ethidium bro-mide to stain oligonucleotides is poor andvariable depending on sequence and com-position.

For more information, see Sewall, A.,Natarajan, P., and Fox, D.K. (1999) FOCUS21, 2.

Analysis of oligonucleotides by absor-bance spectroscopy

Properties that are sequence dependentin oligonucleotides include extinction coef-ficient and A260/280 ratio. • For accurate determination of oligonu-

cleotide concentration, calculate theextinction coefficient using an equationthat incorporates the contribution ofeach base and the effect of base stacking[Newton, P.R. (1995) PCR EssentialData, John Wiley and Sons, New York,New York. p.55.]

• For oligonucleotides, the A260/280 ratio isnot an indicator of purity because theratio of a given oligonucleotide is highlysequence dependent. The reason for thisis the large differences in extinction coef-ficients of individual nucleotides.

For more information, see Fox, D.(1998) FOCUS 20, 84.

Guidelines for primer design for PCRThe ideal PCR primer pair anneals to

unique sequences that flank the target andnot to other sequences in the sample.Poorly designed primers may amplify other,nontarget sequences. The following guide-lines describe the desirable characteristics ofa primer sequence:• Typical primers are 18 to 24 nucleotides.• Select primers that are 40% to 60% GC

or mirror the GC content of the tem-plate.

• Avoid complementary sequences at the3´ end of primer pairs.

• Avoid a GC-rich 3´ end.• Design primers with G or C residues in

the 5´ and central regions.• Avoid mismatches with the target at the

3´ end.• Avoid sequences with the potential to

form internal secondary structure.

Annealing temperature for PCR primersAn important parameter for primers is

the melting temperature Tm. This is thetemperature at which 50% of the primerand its complementary sequence arepresent in a duplex DNA molecule. TheTm is necessary to establish an annealingtemperature for PCR. Reasonable annealingtemperatures range from 55°C to 70°C.Annealing temperatures are generally about5°C below the Tm of the primers. Sincemost formulas provide an estimatedTm value, the annealing temperature is onlya starting point. Specificity for PCR can beincreased by analyzing several reactionswith increasingly higher annealing temper-atures. FOCUS



To learn the latest in PCR techniques,enroll in one of our PCR Techniques orAdvanced PCR Techniques courses at the Life Technologies Training Center.See inside front cover for the schedule.

a g r i c u l t u r a l b i o t e c h n o l o g y


While Chile was declared free ofthe med fly in 1995, nativepopulations of Ceratitis capitataremain in neighboring coun-

tries, so surveillance of over 2,000 miles ofborder is needed. Identification of thegenetic origin of accidentally introducedindividuals would help to localize controland facilitate the surveillance.

Amplified Fragment Length Polymor-phism (AFLP) analysis is an efficient DNAfingerprinting method based on selectiveamplification using PCR of restrictionfragments from a total digest of genomicDNA. The AFLP technique was developedprimarily to reveal the differences betweencultivars of plant species (1) and has beenapplied to several crops. AFLP analysis hasidentified a larger number of molecularmarkers than RAPDs and RFLP in soybean(2) and cotton (3), among other crops. TheAFLP technique has been used to analyzegenetic polymorphism of nematodes (4−5),fungi (6), corals (7), fish (8), and humans (9).One report related to AFLP analysis ofarthropod species has been published (10).Polymorphism of wild populations of C.capitata has been studied using multilocusenzyme electrophoresis and RAPDs (11−13).In this study, the AFLP technique was usedto detect genetic polymorphism in C. capitata(Wiedemann) (Diptera: Tephritidae).

METHODSMATERIALS. C. capitata from strains

Seibersdorf-6096 (S-6), Vienna-60 (a whitepupae temperature-sensitive mutant of S-6),Toliman, and Lluta were obtained fromCentro de Producción de Insectos Estériles,C.P.I.E. from the Servicio Agrícola yGanadero, SAG) located in Lluta, Chile.Toliman is a cross between a native strainfrom Guatemala (Toliman) and the tem-perature-sensitive strain Vienna-42 that hasbeen reared at C.P.I.E. since 1996. Lluta

strain is a cross of native flies collected invalleys of southern Peru and Azapa (north-ern Chile) and has been reared at C.P.I.E.since 1993.

DNA EXTRACTION. Genomic DNA wasprepared from individual larvae or adultspecimens. Each individual conserved in95% ethanol at −20°C was dried undervacuum in a 1.5-ml tube and ground atroom temperature in 200 µl of lysis buffer[50 mM Tris-HCl (pH 8.0), 50 mMEDTA, 3% w/v SDS, 0.1 M 2-mercap-toethanol] with 25 µl proteinase K(10 mg/ml) until the solution turned areddish color. Another 200 µl of lysis bufferwere added, and the mixture incubated for10 min at 65°C. 100 µl of 5 M potassiumacetate were added, and the mixture wasincubated on ice for 10 min. After centrifu-gation for 10 min at 12,000 × g, the super-natant was extracted with 1 volume ofphenol:chloroform:isoamyl alcohol (25:24:1).0.6 volumes of cold isopropanol was addedto the supernatant and incubated at −20°Cfor 20 min. The DNA was collected bycentrifugation, and the pellet was washedwith 70% ethanol, dried, and dissolved in50 µl TE buffer [10 mM Tris-HCl, 1 mMEDTA (pH 8.0)]. DNA was incubatedwith 10 µl RNase A (10 mg/ml) for 45 minat 37°C and then treated with 10 µl ofproteinase K (10 mg/ml) for 15 min at50°C. TE was added to 400 µl, and thesample was extracted with 1 volume of phe-nol:chloroform:isoamyl alcohol (25:24:1).The DNA precipitated with 1/10 volumeof 3 M sodium acetate and 2 volumes ofcold ethanol. After centrifugation, theDNA pellet was washed in 70% ethanol,dried, and dissolved in 40 µl of TE buffer.

AFLP REACTIONS. AFLP products weregenerated using the GIBCO BRL® AFLPAnalysis System I according to the manu-facturer’s instructions. GIBCO BRL TaqDNA Polymerase was used for PCR. The

AFLP products (5 µl) were separated on5.8% or 6.0% (w/v) acrylamide gels at 70to 80 W (1,700 −1,800 V) in 1X TBE on aModel S2001 gel electrophoresis system.After electrophoresis, the gel was dried andexposed to a Kodak X-Omat AR film.

ISOLATION AND CLONING OF POLYMORPHIC GENETIC

MARKERS. The selected bands were cutdirectly from the dry gel, submerged in TEbuffer containing 1 M NaCl, and incu-bated overnight at 37°C. The solution wascollected and ethanol precipitated at−20°C for 30 min. After centrifugation at12,000 × g for 20 min, the DNA wasvacuum dried and dissolved in 20 µl of steriledouble-distilled water. The sample wasamplified using the same pair of primersselected from the AFLP reaction. PCR was35 cycles of 94°C for 30 s, 56°C for 30 s,72°C for 1 min, and a final soak at 4°C.The amplified DNA was visualized on a3% agarose gel stained with ethidium bro-mide. The DNA was ethanol precipitated,and the pellet was washed with 70%ethanol, dried, and dissolved in sterile water.The DNA was ligated to a vector and trans-formed into DH5αF´™ competent cells.Both strands of the clones were sequencedwith the GIBCO BRL dsDNA CycleSequencing System. Sequences were ana-lyzed using the BLAST program from NCBI.

Gino Corsini, Augusto Manubens, Manuel Lladser,Sergio Lobos, and Daniela Seelenfreund

Department of Biochemistry and Molecular BiologyFacultad de Ciencias Químicas y Farmacéuticas

Universidad de ChileCasilla 174 correo 22, Santiago, Chile

[email protected]

Carlos LobosPrograma Nacional de la Mosca de la Fruta

Departamento de Protección AgrícolaServicio Agrícola y Ganadero (Agriculture and Livestock Service)

Bulnes 140, Santiago, Chile

AFLP ™Analysis of the Fruit FlyCeratitis capitata

FIGURE 1. Purified genomic DNA. DNA was extracted from indi-vidual adult flies or larvae from different strains, and 1/4 of thesamples were electrophoresed on 0.8% agarose TBE gels. Lanes 1–3.Strain Lluta (male, female, and larva, respectively). Lane 4. Strain S-6(larva). Lanes 5 and 6. Strain Toliman (males), digested with EcoR I(lane 5) or undigested (lane 6). Lane M. λ DNA/Hind III fragments.

1 2 3 4 M 5 6


a g r i c u l t u r a l b i o t e c h n o l o g y

RESULTS AND DISCUSSIONPurified genomic DNA from an adult

fly or larvae yielded 4 to 5 µg of unshearedDNA with a 260/280 ratio of 1.6 to 1.8(figure 1). In initial AFLP experiments, 2similar strains were screened with 64 primercombinations for at least one differentiatingband (data not shown). Generally, primerpairs produced 35 to 50 bands from 75 to500 bp, but few primer pairs produceddistinctive patterns.

Based on the initial screening, 8 primerpairs were chosen to analyze both sexes of4 laboratory strains. The primer pairE-AAG/M-CAT identified specific markersof Lluta, Toliman, and Vienna-60 in bothmale and female individuals (figure 2). S-6presented a characteristic marker withE-AGG/M-CAG primers (data not shown).

The AFLP patterns from several DNAsamples of the same individual were repro-ducible (data not shown). However, fliesbelonging to the same strain exhibited anumber of bands that varied between indi-viduals. Therefore, our analysis was focusedon identifying marker bands that werecommon for all individuals of a specificstrain. For example, comparing individualsfrom the Lluta strain, the expected com-mon marker band was seen with bothprimer pairs, as well as a similarity in thebanding pattern (figure 3). However, anumber of bands reflecting individual dif-ferences (and high genetic diversity) wereobserved, even though this strain has beenreared in the laboratory for >30 genera-tions. These assays were performed for theother strains, and the presence of therespective marker bands was detected forToliman and Vienna-60 (data not shown).

To use the marker bands as specificprobes, we have isolated and cloned thegenetic marker bands from Vienna-60,Lluta, and Toliman. The fragments were280 bp (Lluta), 360 bp (Vienna 60), and

210 bp (Toliman). All fragments were A/Trich (data not shown). A search of GEN-BANK for these fragments did not matchwith sequenced genes from Drosophila orCeratitis. PCR and Southern hybridizationare in progress to verify that these markerbands can be used as specific probes.

In summary, we have applied the AFLPtechnique to identify laboratory strains ofmed flies. Our goal is to map the geneticdiversity of native wild med fly populationsfrom different geographical locations sur-rounding the Chilean borders. Preliminaryanalysis of wild populations from differentgeographical locations suggests that localpatterns can be obtained. The feasibility ofisolating specific markers for each straincould eventually facilitate the analysis andmonitoring of fly populations.

ACKNOWLEDGEMENTSThis work was financed by the joint

project FAC-SAG/PA-001/97 fromthe Facultad de Ciencias Químicas yFarmacéuticas, Universidad de Chile, andthe Servicio Agrícola y Ganadero.

REFERENCES1. Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van der Lee, T.,

Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., andZabeau, M. (1995) Nucl. Acid Res. 23, 4407.

2. Lin, J.J., Kuo, J., Ma, J., Saunders, J.A., Beard, H.S.,MacDonald, M.H., Kenworthy, W., Ude, G.N., and Matthews,B.F. (1996) Plant Mol. Biol. Rep. 14, 156.

3. Feng, X., Saha, S., and Soliman, K. (1997) Focus 19, 11.4. Roos, M.H., Hoekstra, R., Plas, M.E., Otsen, M., and Lenstra,

J.A. (1998) J. Helminthol. 72, 291.5. Semblat, J.P., Wajnberg, E., Dalmasso, A., Abad, P., and

Castagnone-Sereno, P. (1998) Mol. Ecol. 7, 119.6. Mueller, V.G., Lipari, S.E., and Milgroom, M.G. (1996) Mol.

Ecol. 5, 119.7. Lopez, J.V., Kersanach, R., Rehner, S.A., and Knowlton, N.

(1999) Biol. Bull. 196, 80.8. Liu, Z., Nichols, A., Li, P., and Dunham, R.A. (1998) Mol. Gen.

Genet. 258, 260.9. Schreiner, T., Prochnow-Calzia, H., Maccari, B., Erne, E.,

Kinzler, I., Wolpl, A., and Wiesneth, M. (1996) J. Immunol.Methods 196, 93.

10. Reineke, A., Karlovsky, P., and Zebitz, C.P. (1998) Insect Mol.Biol. 7, 95.

11. Baruffi, L., Damiani, G., Gugliemino, C.R., Bandi, C.,Malacrida, A.R., and Gasperi, G. (1995) Heredity 74, 425.

12. Haymer, D.S. and McInnis, D.O. (1994) Genome 37, 244.13. Sonvico, A., Manso, F., and Quesada-Allue, L.A. (1996) J.

Econ. Entomol. 89, 1208.

FOCUS

FIGURE 3. Presence of marker band in strain Lluta individuals. 5males (lanes 1−5) and 5 females (lanes 6−10) were analyzed withE-AAG/M-CAT (panel A) and E-AAC/M-CAA (panel B). Arrowsindicate the presence of the characteristic marker band.

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 1025 b

p DN

A La

dder

A B

FIGURE 2. Comparison of AFLP patterns. Strains were analyzedusing the selective primer pair E-AAG/M-CAT. Lanes 1 and 2 corre-spond to the analysis of larval tissue from strains S-6 and Lluta,respectively. All other lanes show DNA extracted from adult flies.Lane 3. Strain Lluta. Lane 4. Strain S-6. Lane 5. Strain Toliman. Lane 6.Strain Vienna 60. For each strain, AFLP patterns from one male (�)and one female (�) are shown. Arrows indicate characteristic markerbands for each strain using this primer pair.

1 2 �� 25 b

p DN

A La

dder

3 4 5 6

bp

– 400

– 300

– 200

– 125

m o l e c u l a r b i o l o g y : h e l p f u l t i p s


Saving time and reagents are objec-tives shared by many molecularbiologists. If 2 enzymes can be usedin the same reaction buffer, it could

mean using both enzymes at the same timeor at the very least it could save the timeand resources associated with purificationbetween the reactions. This piece addressesmany commonly asked questions related tobuffer compatibility.

How do I choose a REACT® buffer for my doubledigest with restriction endonucleases?

GIBCO BRL® Restriction Endonucleasesare provided with a REACT Buffer toensure optimal performance. We do notrecommend double digests because ofpossible buffer incompatibility problemsand enzyme steric hindrance problems.However, we know many researchers per-form double digests to save time. Choosethe combination of buffers that yields thehighest activity for both enzymes. It is pos-sible to use a REACT buffer that gives lessthan 100% enzyme activity for a particularrestriction endonuclease and increase theamount of enzyme used in the reaction.Enzyme buffer activity charts can be foundon pages R-52 and R-53 in the 2000–2001GIBCO BRL Catalogue, or search for dou-ble digests in the TECH-ONLINESM sectionof our web site.

Is there a simplified protocol for using alkalinephosphatase that does not require phenolextraction and ethanol precipitation afterrestriction endonuclease digestion?

Yes. In fact, Technical Bulletin 18009-1describes the activity of calf intestinal alkalinephosphatase, bacterial alkaline phosphatase,and temperature-sensitive alkaline phos-phatase in 6 common restriction endonu-clease buffers. If enough of each enzyme(1 unit, 150 units, and 1 unit, respectively)is used, 100% enzyme activity is seen in

REACT buffers 1, 2, 3, 4, 6, or 10.Additionally, it describes a protocol wherecalf intestinal alkaline phosphatase canbe added directly at the beginning of arestriction digest using the REACT buffersmentioned above. The reaction must beincubated for at least 1 h. The bulletin canbe found in the TECH-ONLINE section ofour web site in the Molecular Biologymanuals and technical bulletin section.

Can T4 DNA ligase be used in any REACT buffer?No. T4 DNA ligase comes with a 5X

T4 DNA ligase buffer [250 mM Tris-HCl(pH 7.6), 50 mM MgCl2, 5 mM ATP,5 mM DTT, 25% polyethylene glycol].None of the REACT buffers are comparableto the ligase buffer. The REACT bufferslack the ATP necessary for the ligasereaction. The 5X T4 DNA Ligase Buffer isavailable separately.

What is the activity of restriction endonucle-ases in PCR buffer?

Although common PCR buffer [Tris-HCl (pH 8.4), MgCl2, and KCl) wouldsupport restriction endonuclease activity,we do not suggest adding restrictionendonucleases directly to PCR products.Even though restriction endonuclease reac-tions are done at lower temperatures thanTaq DNA polymerase reactions, Taq DNApolymerase is still active at these tempera-tures. This means that 5´ overhangs generatedby restriction endonucleases will be filled inby the Taq DNA polymerase. Therefore,the best approach would be to clean up thePCR using a product such as theCONCERT™ Rapid PCR PurificationSystem prior to the digestion [see FOCUS(1999) 21, 11]. Alternatively, the use ofTAQUENCH™ PCR Cloning Enhancerwill inhibit the Taq DNA polymeraseactivity during the restriction digestion [seeFOCUS (1998) 20, 15].

Can you use a PCR buffer with DNase I?Yes. DNase I does not come with a

buffer, so the Taq 10X PCR buffer is analternative. Amplification Grade DNase Idoes come with a 10X reaction buffer[200 mM Tris-HCl (pH 8.3), 500 mM KCl,and 20 mM MgCl2]. The Taq 10X PCRbuffer and the Amplification Grade DNase Ibuffer have the same formulation. Thebuffers of the specialized PCR enzymeslike ELONGASE® Enzyme Mix andPLATINUM® Pfx DNA Polymerase have notbeen tested with DNase I.

Since most primers are purchased without 5´ phosphates, can T4 polynucleotide kinasebe added to PCR?

At best, T4 polynucleotide kinasewould show partial activity. The pH ofmost PCR buffers is slightly above theoptimum pH range of 7.4 to 8.0 for theforward kinase reaction. The MgCl2 is lowerthan the suggested optimum of 10 mM.Additionally, the accumulated pyrophos-phate from the PCR would further reducethe kinase activity. The remaining dNTPsfrom the PCR could, however, serve as asubstrate for the kinase. If the objective,however, is to get significant phosphorylation,this approach is strongly discouraged.

Can T4 DNA polymerase be added to thesecond-strand cDNA synthesis reaction topolish the cDNA ends?

Yes. In fact, in all of the SUPERSCRIPT®

Systems for cDNA Synthesis, 10 units ofT4 DNA polymerase are added directly tothe second-strand synthesis reaction. Thecomposition of second-strand synthesisreaction is 25 mM Tris-HCl (pH 7.5),100 mM KCl, 5 mM MgCl2, 10 mM(NH4)SO4, 0.15 mM β-NAD+, and1.2 mM DTT.

Joe Crouse, Teresa Myers, and Julie Brent Technical Services

Part of the Technical Support and Training Team Life Technologies, Inc.

Rockville, Maryland 20849Buffer Compatibility forCommon Reactions


m o l e c u l a r b i o l o g y : h e l p f u l t i p s

Are any of my REACT buffers compatible withthe Klenow (Large Fragment DNA Polymerase I)?

Klenow buffer is in essence REACT 2buffer 10X [500 mM Tris-HCl (pH 8.0),100 MgCl2, and 500 mM NaCl]. Beforepolishing the ends of a cut fragment, heatinactivate the restriction endonucleasedigestion for 10 to 15 min at 65°C. Forenzymes that are not heat sensitive, do aphenol-chloroform extraction. To find outhow active your restriction endonuclease isin REACT 2 buffer, check the referencesection in the Life Technologies 2000–2001Catalogue (page R-52).

What is the best buffer to use to avoid anethanol precipitation step between a ligationand kinase reaction?

Oftentimes, when cloning adapters areadded to fragments to be subcloned, onlyone end is phosphorylated. This means thatonce the fragment is ligated to the adapters,5´ phosphates will need to be added to theresulting product. T4 polynucleotide kinasewill show some activity in PEG-containingT4 DNA ligase buffers. In the SUPERSCRIPTChoice System for cDNA Synthesis, how-ever, the use of a unique 5X Adapter Buffer[330 mM Tris-HCl (pH 7.6), 50 mMMgCl2, 5 mM ATP] is recommended forthe adapter ligation reaction and the phos-phorylation reaction. The ligation of theadapters is done in 1X buffer at 16°C. Afterheat inactivating the T4 DNA ligase at70°C for 10 min, T4 polynucleotidekinase is added to this reaction for thekinase step. FOCUS

“I study the biomolecular mechanisms of vitamin C deficiency diseases.I like to think of myself as a Ricket Scientist.”

c e l l c u l t u r e : h e l p f u l t i p s


p l a n t b i o t e c h n o l o g y

To maximize success, the in vitro cul-ture conditions are set up to mimicin vivo conditions of temperature,

pH, osmolality, and nutrition. Cell culturemedium maintains pH and osmolalityessential for viability and provides thenutrients and energy required for cellgrowth. Basal cell culture media minimallyconsist of salts, amino acids, vitamins, andenergy sources. This paper discusses thefunctions of these components of basalmedia.

PH AND OSMOTIC PRESSUREThe salts in basal medium are primarily

responsible for providing physiological pHand osmotic pressure, membrane potential,enzyme cofactors, and cell attachment.Culture media are buffered to compensatefor the cellular production of CO2 and lacticacid as metabolic by-products. Traditionally,basal cell culture media have been bufferedby bicarbonate (HCO3). As cells grow, CO2

is evolved. The dissolved CO2 forms abuffering system with the bicarbonate.However, if cell density is low or cells haveentered into a lag phase, they may notproduce sufficient CO2 to maintain optimalpH. To counter these potential problems,bicarbonate-buffered media require theuse of incubators with an atmosphere of5% to 10% CO2. Media with low levelsof bicarbonate, such as MEM (1.5 to2.2 g/L), require ∼5% CO2; media likeD-MEM (3.7 g/L), with higher levels ofbicarbonate, require ∼10% CO2 to maintaincorrect pH. The important factor is usingthe correct percent CO2 based upon amedium’s bicarbonate level to maintainphysiological pH, irrespective of cell type.

ENERGY SOURCESCarbohydrates, primarily D-glucose,

and the amino acid L-glutamine are themajor carbon and energy sources in media.Cultured cells have two primary paths ofenergy (ATP) production: conversion ofglucose to lactate (or full oxidation to CO2)and oxidation of L-glutamine to CO2 andammonia.

Traditional glucose levels in culturemedia range from 1 to 4.5 g/L. Generally,the metabolic rate of a cell line correlates tothe optimal glucose level. A slow-growingcell line (long doubling time) will grow inlow or high glucose. However, a fast-growingcell line requires higher glucose levels tomaintain its metabolic rate. Exposure tolower-than-optimal glucose levels couldinduce the cells to enter a lag phase.

A significant portion of the energyrequired to maintain cell growth (∼30%of the energy produced; this can vary withcell type) comes from the oxidation ofL-glutamine. Typical ranges for L-gluta-mine in formulations are 1 to 4 mM, ∼10times the concentration of other aminoacids, due to L-glutamine’s importance asan energy source as well as its role in pro-tein synthesis. The high L-glutamine con-centration present in formulations may alsobe due to its somewhat unstable naturein solution. To counter this, a dipeptideform of L-glutamine (GLUTAMAX™ I,L-alanyl-L-glutamine; GLUTAMAX II,glycyl-L-glutamine) can be substituted forL-glutamine in medium. These dipeptidesare extremely stable in solution and can beautoclaved without decomposition.

Another energy/carbon source that ispresent in media is pyruvate. In carbohy-drate catabolism (glycolysis), D-glucose isconverted via several reactions to pyruvate.Pyruvate can be oxidized further to produceenergy and CO2 or converted to lactate.

NUTRIENTSAmino acids are incorporated into

proteins. At a minimum, basal mediummust contain the essential amino acids—those amino acids that cannot be synthe-sized at a rate adequate to meet metabolicrequirements. More specialized mediumformulations, developed for low serumsupplementation or high cell density, oftenhave non-essential amino acids added toensure that amino acids do not limit themaximum cell concentration attainable.

Vitamins are needed for cell growth andmultiplication. Vitamins are precursors ofenzymatic cofactors. Vitamin concentrationis important in terms of cell survival andgrowth rate rather than directly affectingmaximum cell density.

For most applications, basal mediagenerally require serum supplementation.Serum is largely undefined, but it suppliesessential growth factors and hormonesrequired for cell growth. Serum also providesan additional source of carbohydrates,amino acids, and vitamins. Therefore, serumsupplementation lessens the concerns abouta basal medium choice and whether aspecific formulation provides the essentiallevels of carbohydrates, amino acids, andvitamins. The growth-promoting factorspresent in serum provide a “mechanism” toeasily adapt cells from one formulationto another. In a serum-supplemented appli-cation, choice of basal medium may be sec-ondary to using the “right” serum. FOCUS

Kevin GradyTechnical Services

Part of the Technical Support and Training TeamLife Technologies, Inc.

Grand Island, New York 14072How Basal Media Provide theOptimal Environment for Cell Culture

To learn more about cell culture, enroll inour Cell Culture Techniques or AdvancedCell Culture Techniques courses at theLife Technologies Training Center. Seeinside front cover for the schedule.

transfection invitrogen

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