I’m hoping to get some help with some issues using SapTrap to generate our CRISPR repair templates.
Originally we’ve set up the SapTrap reaction per the protocol, and we’ve had to screen about 40 colonies via colony PCR just go find a few “positive” clones (~4). Then we run the restriction digest of those, and we found out that a majority don’t give the expected banding pattern.
When looking back at the the troubleshooting tips via Dan Dickinson’s CRISPR webpage. One of the suggestions was to run a PCR using the homology primers of the SapTrap reaction which we did and we found a lot of lower bands that are not at the correct molecular weight. But we also saw a band that was at the correct molecular weight. I gel excised that band, and then reset up the sap trap using that band and the backbone.
After transforming and plating we got a good number of colonies. I screen 48 colonies via colony PCR and I got no bands at the correct size. I just got back a bunch of lower molecular weight bands none higher than 3kb, our expected band should be ~7kb.
At this point, I’m not sure what’s going wrong. We thought that if we gel excised the one correct band and then redid the SapTrap we should be getting half of the backbone back and half of the correctly sized band. But that wasn’t the case.
We’ve checked and there are no Sap1 sites in our homology arms.
Thanks for any help anyone can provide!