Superscript® Full-Length cDNA Library Construction Kit - FAQs

The SuperScript Full length cDNA Library Construction Kit has been discontinued. The alternative is the CloneMiner II cDNA Library Construction Kit, Cat. No. A11180.

The following protocol can be used to prepare ssDNA from DH12S or DH5aF', DH5aF'IQ, DH11S cells (this strain not currently offered for sale). Use a stock of the helper phage M13KO7 (1) that is of known titer. For convenience, DH12S cells are supplied with M13KO7 helper phage. DH12S cells are both endA positive, so single stranded DNA isolated from these strains tend to be less contaminated with double stranded rf DNA.

Small-Scale Preparation of ss Phagemid DNA:
1. Pick a single colony of cells containing phagemid DNA and resuspend in 2 mL TBG (1.2% tryptone, 2.4% yeast extract 0.4% glycerol, 17 mM KH2PO4 and 55 mM KH2PO4 and 20 mM glucose) containing 100 µg/mL ampicillin in a 15 mL tube.
2. Immediately add 10 µL M13KO7 helper phage stock at 10E11 pfu/mL.
3. Incubate cells at 37 degrees C with vigorous agitation (275 rpm) for 2 hrs.
4. Add kanamycin to a final concentration of 75 µg/mL and incubate cells at 37 degrees C with vigorous agitation (275 rpm) for 18 to 24 hrs.
5. Transfer 1.5 mL of culture to a sterile microcentrifuge tube and pellet cells by centrifuging at 14,000 x g for 10 min at 4 degrees C.
6. Transfer supernatant to fresh microcentrifuge tube and repeat the centrifugation.
7. Transfer 1.2 mL supernatant to a fresh microcentrifuge tube and add 300 µL of 2.5 M NaCl in 40% PEG 4000.
8. Vortex and incubate on ice for 15 min.
9. Centrifuge at 14,000 x g for 15 min at 4 degrees C.
10. Resuspend the pellet in 50 µL TE and phenol extract to remove the viral coat.
11. Use 10 µL of the final 50 µL volume for gel analysis.
This protocol yields 0.5 to 1 µg ss phagemid DNA.

Large-Scale Preparation of ss Phagemid DNA:
1. Resuspend a single colony in 5 mL of TB or LB broth containing 100 µg/mL ampicillin in a 15-mL tube.
2. Shake at 37 degrees C and 275 rpm overnight.
3. Add 100 µL of the overnight culture to 200 mL LB broth and 100 µg/mL ampicillin in a 1 L flask. Incubate at 37 degrees C with shaking (275 rpm) for 3 hrs.
4. Add 200 µL of M13KO7 helper phage (1 x 10E11 pfu/mL) to the culture and continue to incubate for 2 hrs.
5. Add 1.5 mL of 1% (w/v) kanamycin to the cells for a final concentration of 75 µg/mL. Incubate the infected cells for an additional 18 to 24 h at 37 degrees C.
6. Centrifuge this culture at 16,000 x g for 15 min at 4 degrees C .
7. Filter the supernatant through a 0.2 µm sterile filter into an autoclaved centrifuge bottle. Add 40 µL of DNase I (50 units/µL) and incubate at room temperature for 3 hrs. This step should remove any residual ds plasmid DNA contamination.
8. Transfer 100 mL of the supernatant to another centrifuge bottle. Add 25 mL of 2.5 M NaCl in 40% PEG 4000 to each of the centrifuge bottles containing the supernatant.
9. Vortex the mixture, incubate on ice for 1 h, and centrifuge at 16,000 x g for 20 min at 4 degrees C.
10. Carefully discard the supernatant. To fully drain off the remaining solution from the pellets, place the bottles on an angle, with the pellet side facing up for 10 to 15 min. Remove the solution with a sterile Pasteur pipet.
11. Resuspend the pellets in 2 mL of TE buffer. Add 10 µL of proteinase K solution (20 mg/mL), 20 µL of 10% SDS, and incubate this mixture at 45 degrees C for 1 hr.
12. Transfer the digested mixture to three microcentrifuge tubes and extract four times with an equal volume of phenol:chloroform: isoamyl alcohol (25:24:1), precipitate with ethanol, and dissolve in 100 µL TE buffer.
13. Freeze the solubilized DNA at -20 degrees C for 1 hr and centrifuge in a microcentrifuge at 14,000 x g for 15 min at 4 degrees C.
14. Transfer the supernatant containing the ss plasmid DNA to a fresh tube and discard the polysaccharide pellet. Store the ssDNA at 4 degrees C.
15. Determine the DNA concentration (OD260).
This protocol yields ~100 to 200 µg ss phagemid DNA.

(1) Vieira, J. and Messing, J. (1987) Methods in Enzymology 153, 3.

Lawn cells for M13 infection must be made with an E. coli strain containing the F' episome. Some of our competent cells that may be used include: DH5aF'IQ, INValphaF', TOP10F', OmniMAX2, Stbl4, or DH12S cells.

To make a glycerol stock of lawn cells:
1) Streak cells on an LB plate and incubate overnight at 37C.
2) Pick a single colony from the plate into 50 ml LB or S.O.B. medium and incubate the culture at 37C until the OD550 reaches 2.0.
3) Centrifuge the cells in a clinical centrifuge at 3,000 x g for 10 min at 4C.
4) Resuspend the pellet in 5 ml 60% S.O.B. and 40% glycerol.
5) Aliquot the cells and freeze them in a dry ice/ethanol bath. Store the cells at -70C.

LB Medium (per liter):
- 10 g Tryptone (SELECT Peptone 140)
- 5 g Yeast Extract (SELECT Yeast Extract)
- 10 g NaCl
Add Distilled Water to a final volume of 1 L. Stir to dissolve, and autoclave.

S.O.B. Medium (per liter):
- 20 g Tryptone (SELECT Peptone 140)
- 5 g Yeast Extract (SELECT Yeast Extract)
- 10 ml 1 M NaCl - 2.5 ml 1 M KCl - 10 ml 1 M MgCl2 - 10 ml 1 M MgSO4
Add tryptone, yeast extract, NaCl, and KCl to a flask or bottle, and add Distilled Water to a final volume of 1 L (~980 ml). Stir to dissolve, autoclave, and cool to room temperature. Then add Mg++ stock solutions (1 M MgCl2/6H2O and 1 M MgSO4/7H2O, filter sterilized). Mg++ solutions should not be added before autoclaving, as this will likely result in precipitation.

Plasmid libraries may be amplified using several different methods. Growth in liquid culture will frequently result in skewed representation of clones due to differential growth characteristics of individual clones. To minimize these representational biases, we recommend semi-solid amplification. With this method, colonies are grown in 2X LB broth and 0.3% agarose in suspension, facilitating equal growth of all clones and avoiding disproportionate amplification.

Alternatively, amplification of plasmid libraries may be done on selective agar plates, as described below. Libraries containing expression vector plasmids such as pSPORT or pCMV SPORT should be grown under noninduced conditions (no lactose, no IPTG). The lac repressor gene encoded on the pSPORT plasmid is sufficient to prevent expression of the recombinant genes cloned into the multiple cloning site. Repression of expression is necessary to ensure the amplification of clones whose gene products may be harmful to survival of the E. coli host.

Recommended protocol for Library Amplification on Plates:
1. After titering the original library, plate the cells at a density of ~1 x 10E4 CFU per 100 mm plate or ~0.5 x 10E5 CFU per 150 mm plate. Use selective media containing the appropriate antibiotic.
2. Grow at 37°C until a thick lawn is visible (~6 to 12 h).
3. Add 5 ml per 100 mm plate or 15 ml per 150 mm plate of S.O.C. medium or LB broth.
4. Gently coax the bacterial lawn into the broth with a cell scraper (or a glass slide).
5. Collect the suspension and repeat steps 3 and 4 once more. Pool the broths, then incubate at 37°C for no longer than 1 h with constant swirling.
6a. If the bacterial suspension is highly dense, add glycerol to a concentration of 15% to 50%; aliquot the cells into cryovials; and immediately store them at -70°C.
6b. If the bacterial solution is too dilute, centrifuge at 7,000 x g for 10 min and resuspend in a smaller volume of 15% to 50% glycerol/media and freeze.
7. For use, thaw the cryovials and plate dilutions onto selective media.

NOTE:
-The viability of E. coli stored at -70°C will not change substantially for years, unless thawed and refrozen.

It depends on whether the lambda vector has recombination (att) sites that are compatible with the design of our system. Without att sites, the library would definitely not be Gateway compatible.

Yes, the Gateway LR reaction allows you to transfer a cDNA library made in Gateway entry vectors into Gateway destination vectors with no significant effect on the library's average insert size or insert size range. Thus, the library's complexity is maintained after the LR reaction. The library transfer reaction protocol is found in the CloneMiner II cDNA Library Construction Kit manual.

There is no recommended method that would maintain the complexity of the original library. The best option is to create a new library using the CloneMiner II cDNA Library Construction Kit. This kit will allow you to clone, via a BP reaction, all the generated cDNAs into a Gateway-compatible vector.

Columns are stored at 4 degrees C. Over time the ethanol can evaporate, leaving behind an air bubble. To correct this, users should wash the columns with one volume of dH2O and then use the "thumb trick". Add the dH2O to the column, if it is still flowing slowly, place a gloved thumb over the top of the column, push down a little, and then raise up (you can do this 2-5 times). Similar to a pump, this helps the flow rate and gets rid of the air bubble that was preventing a good flow. This is the procedure that is used at our training center.

Labeling can be carried out in either reaction, but labeling during first-strand synthesis allows users to detect problems in the RNA preparation or in the first strand components. These problems can be solved before going to the time and expense of the second-strand synthesis.

DNA concentration is a critical parameter in PCR amplification. If too much DNA is used, a smear will often result (due to concatamer formation of the target DNA that can occur at higher concentrations). When reamplifying PCR product, a good starting concentration is 10 pg. If the DNA cannot be quantified, it may be necessary to set up reamplification reactions using serial dilutions of the original product to obtain good amplification of a single-sized product.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.

PCR has been used for random mutagenesis of a specific DNA region. The method for creating these random mutations uses conditions that reduce the fidelity of AmpliTaq DNA Polymerase. Briefly, DNA is amplified in the presence of buffer, manganese, and an unequal molar concentration of dNTPs.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.