Troubleshooting Guide

1. Low yield of the first-strand cDNAs. The success of the synthesis of first- and second-strand cDNAs is extremely important and is considered to be halfway done in constructing a cDNA library. If the yield of the firststrand cDNA conversion is less than 8%, the intensity of the different cDNA sizes shown on the x-ray film is very weak or the size range is very small and of lower molecular weight, these problems are likely caused by poor mRNA preparation or the failure of reverse transcriptase. The quality of the mRNA templates is the most important factor for successful cDNA synthesis. If the poly(A)+RNA is degraded during isolation or purification procedures, the first-strand cDNA synthesized from the template is only partial-length cDNA. To avoid this problem, check the quality of mRNA

templates prior to the synthesis of first-strand cDNAs by analyzing 2 mg mRNAs in 1% agarose-formaldehyde denaturing gel containing ethidium bromide (EtBr). Following electrophoresis, take a photograph of the gel under UV light. If the smear range is from 0.65 up to 10 kb, the integrity of the mRNA is very good for the synthesis of cDNA. Another possible cause is that AMV reverse transcriptase for the synthesis of first-strand cDNAs fails in the middle of the procedure. In that case, the cDNAs produced are likely to be only partial length. Try to use positive mRNA templates as a control to check the activity of reverse transcriptase. If mRNA templates and reverse transcriptase are very good, then use control RNA to check for the presence of inhibitors such as SDS, EDTA, and salts.

2. Low yield of the second-strand cDNAs. A yield of the second-strand DNA conversion of less than 40% or a size range that is very small and of abundant lower molecular weight may be due to RNase H or DNA polymerase I that does not function well. To check the activity of RNase H, treat control mRNA with the enzyme and assayed by electrophoresis. If the RNase H works well, the mRNA is nicked into fragments. This will be indicated by gel electrophoresis as compared with untreated mRNA. On the other hand, if DNA polymerase I does not function well, the synthesis of second-strand cDNAs or the repair of gaps will not be completed, thus generating partial-length cDNAs. In that case, set up another reaction using a control experiment or use fresh DNA polymerase I. If low incorporation occurs even with the positive control, it is likely that the radioisotope used as a labeled probe is not good. Check the specificity and half-life of the isotope and use fresh [a-32P]dCTP as a control.

3. Plaques are small in one area and large in other areas. This is due to uneven distribution of the top agar mixture. Make sure that the top agar mixture is spread evenly.

4. Too many positive clones in the primary screening. The specificity of the primary antibodies is low, the concentration is too high, or the blocking efficiency is low. Try to use IgG or an affinity column-purified antibodies and carry out different dilutions of the antibodies, or increase the percentage of the blocking reagent in the TBST buffer.

5. Purple background on the filter. Color development is too long. Try to stop the color reaction as soon as desired signals appear.

6. Unexpected larger purple spots. Air bubbles may be produced when the filters are overlaid on the LB plates. Make sure that no air bubbles are generated underneath the filters.

7. No signals appear at all in any of the primary screening plates. The pfu numbers used for each plate may be too low. For rare proteins, 2 x 106 pfu should be used in primary screening of the library.

8. Signals are weak on the filters of subsequent screening. The antibodies have low activity or color development was stopped too early. Try to use an immunoassay to check the quality of the antibodies, allow the color development to proceed for a longer time, or try to use high-quality nitrocellulose filters.

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