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DNA Gel purification column issues/mistakes? What's your process?

DNA Zymogen Gel Purification PCR

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#1 kidwolf3

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Posted 20 December 2013 - 12:49 PM

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!



#2 phage434

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Posted 20 December 2013 - 01:00 PM

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.



#3 kidwolf3

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Posted 20 December 2013 - 01:07 PM

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!



#4 phage434

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Posted 20 December 2013 - 01:13 PM

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.



#5 kidwolf3

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Posted 20 December 2013 - 01:41 PM

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)



#6 phage434

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Posted 20 December 2013 - 02:34 PM

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.



#7 christy

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Posted 22 December 2013 - 04:12 PM

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.







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