Special Applicationsin Agarose Gels

Section IX: Special Applications in Agarose Gels

In This Section
Amplification of Plasmid cDNA Libraries withSeaPrep® Agarose
Preparing Agarose for use inCell Culture Applications
References

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Section IX: Special Applications in Agarose Gels
Amplification of Plasmid cDNA Libraries with SeaPrep® Agarose

Introduction
This section describes an amplification techniquedeveloped to address the common problem ofdisproportionate amplification of plasmids seen when
expression cloning mRNAs of very low abundanceencoding cytokines, receptors, and cell surface moleculesfrom plasmid cDNA libraries derived from highly complex
tissue sources. This amplification technique allows
amplification of a plasmid cDNA library in a representative
fashion, decreasing the possibility that less abundant
clones would vanish during the amplification due todifferential rates of replication.
The technique below describes a method wherebacterial transformants are suspended, not plated,in low gelling temperature agarose. With thismethod, it has been found that, from generation
to generation of amplification, the relative abundance ofbacterial cells containing plasmids encoding selected
hematopoietic factors varies less than two fold.

cDNA/vector annealing reactions
1.

Prepare vector using a tailing method.

2.

Prepare and purify cDNA following standard
procedures.

3.

Optimize the tailing of cDNA inserts following standard
procedures.

4.

Scale up the appropriate cDNA/vector-annealing reaction
to generate up to 2.5 x 106 cfu/2000 ml.

5.

Perform the appropriate number of standard bacterialtransformations following standard procedures.

6.

Pool all the transformations after the 37°C incubation.

Note: General procedures for vector and cDNA preparation,ligation and transformation can be found in Sambrook, J., et al.
and Kriegler, M.
■■ Materials–– Autoclave

Preparing the bacterial growth media plus agarose–– 2 L Erlenmeyer flasks

Note: Alternatively, prepared solutions of L Broth, LB Brothor Super Broth can also be used.

–– Water bath set to 37°C

1.

I n a 2 liter Erlenmeyer flask, add the following reagentsto 1 liter of double distilled water:		 1a. 20.0 g bacto-tryptone		

1b. 10.0 g bacto-yeast extract
1c. 10.0 g NaCl–– pH meter–– Sterile 50 ml polypropylene centrifuge tubes–– Ice water bath–– Sterile 500 ml centrifuge bottle–– Centrifuge–– Sterile Nunc® Tubes

Special Applicationsin Agarose Gels

2.

Measure pH of solution.

3.

If necessary, adjust to pH 7.0 using 1 N NaOH or
1 N HCl.

4.

Add 3.0 g of SeaPrep® Agarose to the solution.

5.

Autoclave the solution on a liquid cycle for 15 20 minutes.

6.

Cool the solution to 40°C.

–– Double distilled water

7.

Store in a 37°C water bath until ready-for-use.

–– Bacto-tryptone

8.

Prior to use add 2 ml of 50 mg/ml ampicillin.

–– Bacto-yeast extract–– 0.2 µm filter■■ Reagents–– SeaPrep® Agarose–– 2X L Broth (LB)–– 50 mg/ml ampicillin–– NaCl–– 1 N NaOH or 1 N HCl–– pH standards–– LB-amp plates–– Filter sterilized glycerol

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Section IX: Special Applications in Agarose Gels
Amplification of Plasmid cDNA Libraries with SeaPrep® Agarose — continuedPlasmid cDNA library amplification1.

Add ampicillin to a final concentration of 100 µg/ml(2.0 ml of 50 mg/ml stock solution) to the 2X LB-agarose
solution.

2.

Add transformants (1.25 x 106 cfu) to the LB-agarose
solution.

3.

Gently swirl to avoid foaming.

4.

Aliquot the transformation mix into 25 ml aliquots in
50 ml polypropylene centrifuge tubes.

5.

Library Storage
1.

Prepare 12.5% glycerol by diluting in double distilledwater.

2.

Filter solution through a 0.2 µm filter into a sterile
container.

3.

Pour the colony-containing gel into a sterile 500 ml
centrifuge bottle.

4.

Place the tubes in an ice-water bath for 20 - 60 minutes
to allow the agarose gel to set.

Pellet the cells at 8,000 rpm for 20 minutes at roomtemperature. It is not necessary to melt the gel, the cells
will pellet through the soft agarose.

5.

Decant the media from the cell pellets.

6.

Incubate overnight at 37°C undisturbed.

6.

7.

P late 100 µl of the cell-agarose suspension directly on
LB-amp plates for titer determination or prepare thesuspension for library storage.

Resuspend the cell pellets in 100 ml of 12.5% glycerol in
2X L broth.

7.

Aliquot into sterile Nunc® tubes.

8.

Stores library at ­—70°C.

■■ Materials–– Scalpel or razor blade–– GelBond® Film–– Glass plate or press board–– Clamps or elastic bands–– A forced hot-air oven■■ Reagents–– SeaKem® LE or GTG® Agarose–– SeaKem® Gold Agarose–– NuSieve® 3:1 or NuSieve® GTG® Agarose–– MetaPhor® Agarose

Special Applicationsin Agarose Gels

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References
Kriegler, M., Gene Transfer and Expression: A Laboratory Manual, Stockton
Press, 1990.
Perez, C. and Kriegler, M., Resolutions, 6(4): 1 - 2, 1990.
Ausubel, F.M., et al., Current Protocols in Molecular Biology, Wiley & Sons,
1995.
Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition,
Cold Spring Harbor

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Section IX: Special Applications in Agarose Gels
Preparing Agarose for use in Cell Culture Applications

Introduction
Agarose has unique properties that make it especially
useful in cell culture media applications. Agarose can form
stable gels at concentrations as low as 0.3% (w/v), hasvery low ionic concentrations and is free of contaminating
impurities that may affect the growth characteristics ofcells in culture. Agarose could be used directly without
supplementation for plaque assays ensuring high cellvariability. The use of agarose is suggested for media
where the absence of a nutrient is mandatory.
Advantages–– Highly purified, eliminating contaminants–– Excellent optical transparency which enhancescolony observation–– Gelling temperatures below 28°C allowing
manipulation of cells in solution–– Can be used to produce a ‘defined’ media systemApplications–– Cell culture media–– Mammalian–– Plant–– Bacterial

The concentration and type of agarose to use largely
depends on your cell system, the application, and the lot
specific gel strength of the agarose. There are three typesof agarose which can be used for cell culture media, eachhaving unique properties making one more suitable for agiven application than another.
—— SeaKem® LE Agarose – A standard melting and gelling
temperature agarose which can be used as a solid
medium to support cell growth by supplementation
with growth factors and nutrients, top agar enrichedwith magnesium for baculovirus screening, substrate
for bacterial growth and for colony lifts—— SeaPlaque® Agarose – A low gelling temperature agarose(28°C) that remains a liquid at 37°C allowing themanipulation of cells within the solution; SeaPlaque®
Agarose can be used as a semi-solid media foranchorage independent assays, plaque assays oroverlays; SeaPlaque® Agarose has also been found tobe very effective as a medium for protoplast culture—— SeaPrep® Agarose – Unique ultra-low gelling temperature(15°C) and gel strength agarose (>75 g/cm 2);
SeaPrep® Agarose is ideal for hybridoma cloning;
Cells can be recovered from the gel by increasing thetemperature slightly, allowing transfer to a viable cellsuspension for subsequent growth in liquid medium–– Viral–– Overlay assays–– Plaque hybridization assays–– Soft agarose hybridoma cloning–– Attachment dependent cell culture–– Colony lift assays–– Analysis of matrix and cell matrix interactions

Special Applicationsin Agarose Gels
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Preparing Agarose for use in Cell Culture Applications — continued

Suggested Agarose Products andConcentration Guidelines

Agarose Preparation

The table below provides general guidelines on agaroses
and gel concentrations for given applications. General
guidelines and specific information pertaining toa given cell type and application can be obtained from thecurrent literature and Cell Biology: A Laboratory Manual.

Application
Mammalian cell culture

SeaKem® LE Agarose
0.3% - 1%

When preparing agarose for cell culture work, it isalways best to prepare the agarose in water suitablefor cell culture and separate from any growth media
or nutrients. Agarose solutions and media solutions
should be prepared at 2X concentrations (i.e., if
desired final agarose concentration is 0.6%, prepare a
1.2% agarose solution), autoclaved separately, andaliquoted into useable aliquots.

SeaPlaque® Agarose
0.25% - 1.5%

Baculovirus screening 		
0.3% - 0.6%
Bacterial culture
0.7% - 1.0%
1.0%
Colony lifts
0.7% - 1.0%
1.0%
Anchorage independent assays
0.25% - 0.5%
Plaque assays
0.3% - 1%
0.50% - 1.5%
Overlays
0.3% - 0.6%
0.3% - 1%
Protoplast culture 		
0.35% - 0.7%
Hybridoma cloning 			
Virus enumeration 			Cell matrix interactions 		
1.0% - 3.0%
Attachment dependent cell culture 		
0.6%
In vitro cloning – mammalian
0.5% - 2% 		

SeaPrep® Agarose

0.8% - 1.5%
1%

1% - 4%

 otes: Higher agarose concentration gels can affect and possibly restrict cell proliferation. Optimal gel concentrations should be determined for each cultureNsystem on a case-by-case basis. In some cases, such as hybridoma cloning using SeaPrep® Agarose, it is advisable to sample different lots of agarose for thedesirable gel strength qualities.

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Section IX: Special Applications in Agarose Gels
Preparing the Agarose for Use in Cell Culture Applications — continued

Procedure for Autoclaving Agarose

General Procedure for Using Agarose in Culture Medium

1.

Choose a flask that is 2 - 4 times the volume of thesolution.

1.

Remelt the agarose by placing in a hot water bath ormicrowave.

2.

Add water to the flask.

2.

Allow the agarose solution to cool to 37°C.

3.

Sprinkle in the pre-measured agarose powder at a 2X
final agarose concentration.

3.

Prewarm the 2X media solution to 37°C.

4.

4.

Cover the flask with aluminum foil.

5.

Place the flask in the autoclave.

Mix equal volumes of the sterile 2X agarose solution
with sterile 2X media containing growth factors andnutrients.

6.

Sterilize the agarose by autoclaving for 10 minutes at 15
lb/in2. If using SeaPrep® Agarose, autoclave for no longer
than 5 minutes.

5.

Cast the agarose/media solution into plates or sterileculture tubes.

6.

Allow the agarose solution to gel for 20 minutes if using
as a feeder or overlay or maintain the solution at 37°C if
using as a liquid culture.

Note: Agarose may lose gel strength when exposed to longer

periods of time in the autoclave.
7.

Once the agarose solution has cooled, aliquot intouseable aliquots and store at 4°C prior to use.

■■ Materials–– Flask that is 2-4 times the volume of the solution–– Autoclave–– Aluminum foil–– Sterile flasks or culture tubes■■ Reagents–– Agarose powder–– Water suitable for cell culture

Note: A solution containing 1% SeaPlaque® Agarose will
stay liquid for approximately 18 hours. The amount oftime an agarose solution will stay in a liquid state at 37°C,
largely depends on the agarose concentration (increased
agarose concentrations will decrease the time the solution
stays in a liquid state), the age of the agarose and theparticular lot of agarose you are using. When purchasing
a new lot of agarose, we recommend you test this prior toculturing cells.
■■ Materials–– Microwave or hot water bath■■ Reagents–– Gelled agarose solution–– 2X media solution

Special Applicationsin Agarose Gels

References
Borden, E.C., et al., Appl. Microbiol. 20: 289 - 291, 1970.
Prescott, D.M., Methods in Cell Biology, 1st edition, Academic Press, 1975.
Saunders, F.J.K. and Smith, J.D., Nature 227: 513 - 515, 1970.
Mack, D.O., et al., BioTechniques, 17(5): 860 - 863, 1994.
Buschmann, M.D., et al., J. of Orthopaedic Research, 10: 745 - 758, 1992.
Calba, H., et al., Plant and Soil 178: 67 - 74, 1996.
Shantze, K.A., et al., Cancer Research 45: 4416 - 4421, 1985.
Celis, J.E., Cell Biology: A Laboratory Manual, 2nd edition, Academic Press,1998.
Bagasra, O., et al., Cancer Immunol Immunother, 20: 55 - 60, 1985.
Civin, C.I., et al., J. Immunol. Methods, 61: 1 - 8, 1983.
Buschmann, M.D., et al., J. of Cell Science, 108: 1497 - 1508, 1995.

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