DNA Gel purification column issues/mistakes? What's your process? - (Dec/20/2013 )
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!
I would definitely elute in higher volume, such as 50 ul, or 30 ul twice. Make sure you allow the elution buffer to sit on the column for a few minutes before spinning down.
You should also make doubly sure you do a good dry spin after the final column wash -- this is important to remove ethanol from your final product.
I'm guessing that ethanol comprises a major portion of your 6 ul elution, and that most of your DNA is still on the column.
I hate gel purification, and if you can possibly do it, I would avoid it. But you'll have the same problem if you don't dry spin and elute in a reasonable volume.
I'm guessing you are trying to do ligate at too high a concentration of DNA (why else would you be eluting in 6 ul?). You don't want to do this, in any case. Low concentrations favor circular product, which is what transforms.
I need to also clarify, sorry I forgot = my PCR product I want to transform is LINEAR. Its a yeast homologous recombination....its not a super efficient process, I need HIGH concentration! Thanks again!
Right. I still think 6 ul is too low. And I'd recommend a double elution even then. The first elution will dilute ethanol on the column and partially elute -- the second one will elute with essentially pure elution buffer. You could also try min-elute columns, which are intended for low volume elution. I think the Zymo columns also are set up to elute in low volumes. I wouldn't try with less than 15 ul x 2 on a normal column.
Ok thanks. And for checking my product?
Do you recommend I go straight from Thermocycler to gel? Or take a small sample of my PCR product, and then run on gel to confirm? (then take the rest and purify)
Yes, you can go directly from PCR product (2-5 ul) to a gel. If you get a clean band, I would definitely not do a gel purification. If you don't get a clean band, then I think it might be worthwhile to optimize your PCR reaction rather than messing with gel purification.
If it is PCR product (if it is single band product), directly go to purification. No need of gel purification. you can use the same buffer and you will get concentrated product. I use Promega buffers for direct PCR product purification.