18080-051 - FAQs

A low number of cDNA clones could result due to the reasons listed below:

(1) Phenol extractions were performed with phenol that was equilibrated with water that was not DEPC-treated.

(2) Poor first strand yield.

(3) No RNase H was added to second-strand reaction.

(4) Second-strand reaction was performed at a temperature greater than 16 degrees C.

(5) Dilution of first-strand reaction was made incorrectly: exact dilution is crucial, because the pH of the second-strand reaction differs from that of the first-strand reaction.

(6) The size fractionation column ran too quickly, the column was allowed to dry before fractions were loaded, or the column was not washed thoroughly to remove ethanol.

(7) Competent cells were of poor efficiency: verify efficiency of transformation with a control DNA.

(8) There was an insufficient amount of cDNA in the ligation mix.

(9) If cells were transformed by electroporation, there may have been too much salt in the electroporation mix, which kills cells.

Please see listed below a few reasons why you may be getting small first-strand cDNA products:

(1) Template was degraded by RNase contamination: maintain aseptic conditions.

(2) Secondary structure is present in the RNA:
-Heat the RNA to 70 degrees C for 10 min and quick-chill on ice to denature the RNA. Increase the temperature of the first strand reaction up to 50 degrees C. Denature the RNA by treatment with 20 mM methylmercuric hydroxide (see Krug et. al. (1987) Methods Enzymol 152:316).
-Try alternate reverse transcriptase such as SuperScript III polymerase and perform first-strand synthesis at higher temperature.

(3) Alkaline gel electrophoresis was performed incorrectly: use a 1% alkaline agarose gel.

(4) For random-primed first-strand cDNA, an excessive concentration of primers was used: use 50 ng random hexamers/1-5 µg total RNA.

(4) Incorrect fractions were taken at the column chromatography step.