4.1. No PCR Product

Possible reasons:

1. Incorrect primers or bad reagents used
2. Missing reagents
3. Low transduction of target cells
4. Poor gDNA purification prep contains PCR inhibitors
5. PCR conditions are not optimal

Solutions:

• Include 1 ng of plasmid library DNA as a positive control. If it produces the correct amplification product, the problem lies either with absent or low numbers of sgRNA (e.g., low MOI or problems with the transduction efficiency) or impurities in genomic DNA that block sgRNA amplification. Dilute the genomic DNA 2-5 fold and repeat the amplification step in several test tubes. If the positive control does not produce the correct product, confirm use of the correct primers and reagents.

• Verify that primer sequences are correct.

• If you don’t see a band for any of the samples, we suggest increasing the number of PCR cycles as follows:
  • Run the First Round PCR at 18, 20, and 22 cycles and retrieve 10 µl aliquots at each cycle number. For example, run the first PCR and retrieve 10 µl aliquots at 18 cycles, cycle 2 more times to 20 cycles and retrieve 10 µl, and cycle a couple more times to 22 cycles. Don’t run these on a gel because you are unlikely to see a band—and you don’t want to overcycle them until a band appears.
  • Use 5 µl of each cycle-number aliquot collected during the first PCR. Run the Second Round PCR on each of the first PCR reactions, and then check for a clean visible band. Start with 14 cycles for the second PCR; if you don’t see a band, cycle 3 more times to 18; if no band, 3 more to 21, and then 24.
  • If you see at least a faint band by 25 cycles second PCR, then cycle a couple more times to produce a clean band.
  • If you don’t see any band by 25 cycles of the second PCR, then you can go back to the first PCR and try adding a few more cycles to 26 and 28, and then run the second PCR again up to a max of 25 cycles. If you don’t see any band by then, there is a problem with the samples.
  • if no band is observed with ~40 combined cycles between the two PCR reactions, it is very likely there is likely a more significant problem with the DNA samples or PCR set up.