Positive Control:

Use the original plasmids (pCBC-DT1T2 and pCBC-DT2T3) as positive controls in your PCR reactions. This will help you determine if the problem lies with the primers or the PCR conditions.

Primer Design:

Double-check your primer sequences for any errors. Ensure that the primers are designed correctly based on the target sequences of the guide RNAs.

Verify the primer Tm (melting temperature) and GC content to ensure they are suitable for the PCR conditions you are using.

PCR Conditions:

Adjust the annealing temperature: Since you are using a gradient of 60°C, you may try a temperature optimization step to find the optimal annealing temperature for your specific primers.

Check the extension time: Make sure that the extension time (30 seconds) is sufficient for the expected product size based on your primers.

Master Mix and Reagents:

Ensure that your Platinum SuperFi II Green Master Mix is not expired and has been stored properly.

Consider testing a different batch of master mix or using fresh reagents to rule out any issues related to reagent quality.

PCR Setup:

Be careful with pipetting accuracy and ensure that the correct volumes of template DNA, primers, and master mix are being added to each reaction.

Use separate areas for setting up PCR reactions to avoid contamination.

Troubleshooting PCR Failures:

If none of the above steps resolve the issue, you might consider troubleshooting PCR failures in more detail. This could involve trying different PCR conditions, re-designing primers, or exploring alternative PCR enzymes.

Joseph Ikwu , Thank you for your reply. I have a question regarding primer design. When incorporating the 19-nt gRNA into primers (DT1-F0, DT2-F0, and DT3-BsR), should we add the gRNA sequence in the 5'-3' orientation, or use its reverse complement? Additionally, if our gRNAs are designed in the 3'-5' direction relative to the gene, should we insert the 19-nt sequence in the 3'-5' direction or its reverse complement in the 5'-3' direction?