I am trying to run a PCR to verify insertion of my construct into the AAVS1 locus in iPSC using CRISPR.

I designed three primer pairs to amplify the left and right insertion regions (one binding inside the insert, one binding outside; see image), and one primer pair to amplify a region inside the insert. The insert contains a fluorescent protein, which I can see expressed in the cells under the microscope, so I am pretty sure that the insertion has worked correctly; however, I cannot get any specific PCR product for sequencing. (Even if it has been inserted unspecifically somewhere in the genome, since I am seeing the fluorescent reporter in the cells, I would expect at least to get a positive result for the "internal" region.)

I used DNAzol to purifiy gDNA from the cells, and when I checked it on a gel I noticed two additional bands, which I thought might be rRNA (see image), and when I treated the samples with RNAse the bands disappeared, so I continued happily with the PCR.

For the "internal" region, I am able to use the plasmid as a control, and here I can see a specific PCR product with the expected size, however, for all other plasmid / gDNA template combinations, I get a huge amount of large-sized unspecific PCR products (see second image). Which is why I am currently suspecting that something is not right with the gDNA? But it looks pretty good on a gel.

I am using KOD1 polymerase (KOD1 master mix) according to manufacturer instructions when it comes to amplification from gDNA:

PCR: Total 25 µl per reaction

1.25 µl DMSO

1 µl primer fwd [10 µM]

1 µl primer rev [10 µM]

8.25 µl H2O

12.5 µl KOD master mix

0.5 µl DNA (= 25ng)

Init. Denat. 94°C 1.5 min

Denat. 94°C 5 sec

Anneal 58°C 5 sec

Extension 68°C 1 sec

and also tried

Init. Denat. 94°C 3 min

Denat. 94°C 45 sec

Anneal 58°C 45 sec

Extension 68°C 1 min

I have triple-checked the specificity of the primers, and compared with other primers used in literature for the same purpose (AAVS1 locus). I have re-designed new primers that bind in slighly different places. I have tried different elongation / annealing times and temperatures... It always looks the same (large-size unspecific products).

I a last-ditch effort, I cut out pieces of gel from the "unspecific" results around the size where I would expect the PCR product, and repeated the PCR with those as a template - I got some promising looking results on a gel, but when I sent them for sequencing, it was all unspecific.

I am currently at my wit's end and hope someone else has seen something similar and was able to solve it in the end!!

(Plan B will be to re-do the DNA extraction and try again from the beginning I guess...)