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

Solutions:

  • Include 10 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.
Last modified: May 10, 2018

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