Im running a couple 50 ul PCR reactions and then running the whole volume (+8 ul loading buffer) on gel, cut gel pieces out and purify using the Zymogen gel recover/purify kit (columns and all that).
After all that, my transformations have not come out very well....I suspect I have a problem here with purifying from gel, as my bands are correct product, very nice intensity.
What I am doing so far:
1) I dissolve each gel fragment in 900 ul ADB. Then I continue running multiple (4-5+) gel fragments on a single column...is that a mistake? I believe at some point my column would be saturated and I am actually losing a lot of my DNA by flow-through. Should I use a different column for each gel fragment? At most 2 runs on the same column? Not sure...
2) The protocol calls for eluting in at least 6 ul of buffer or water (I use sigma water). I elute this volume.
3) I got a tip I intend to try out next attempt = vortexing every few minutes while dissolving the gel ( gel + ADB , 15 minutes on heatblock at 55-60 C)
What is your process?
-Do you take a small sample of the PCR product (maybe i'll take a couple microliters out of my 50 ul) and run it first on a gel to test? Then purify the rest of the PCR product directly (without doing a gel purify)?
Thank you all!