If so, how did you get it clean enough to inject into the worm?
We are doing overlap PCR to stitch fragments together AND add homology arms for CRISPR insertion. We get a prominent band at the correct size but also a smaller band of equal intensity. We don’t want to run the risk that the smaller fragment has some of the homology arms (since it amplifies with the outer primers) and would be preferentially inserted into the genome, hence the need to gel extract the correct band.
Thanks,
Steve
try using SPRI magnetic beads and play with the ratios to selectively bind your product. They’re pretty useful for both size selection and also concentrating.
I used them recently for a CRISPR injection because I had ~500uL of PCR reaction that I needed to concentrate for the Melo protocol.
I’ve injected QiaQuick-purified PCR products cut out of gels, for rescue. It worked OK.
yeah, not sure if all the left over salts etc. that end up after column clean-up affect the success of a CRISPR injection if you’re injecting the ALT-R Cas9. my labmates usually just do ethanol precipitation to clean up and concentrate but that’s way more effort than the beads.
Snug - yes we are doing the Mello protocol, so we will give the SPRI beads a shot. A former labmate also asked if I had access to a Pippin Prep machine, which is used in NGS to size-select. She indicated that you get clean DNA with good yield. I like the fact that you can concentrate with the SPRI beads, though. Thanks for all the advice!
Steve