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want to purify PCR product from agarose gel but have primer dimer


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3 replies to this topic

#1 piggle

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Posted 22 August 2009 - 11:42 PM

Hi, :lol:
I've question for my PCR. I want to purify my PCR product (300 bp)from agarose gel for cloning but primer dimer was seen.
Someone told me that I should optimize PCR condition until dimer is disappeared. He said that dimer may be contaminate in my PCR band, so i may get wrong clone.
But, I think that dimer is smaller than my PCR product and its band far from my band, and if I cut PCR band carefully, dimer should not contaminate my PCR product.

Anybody have suggession for me.

Thank advance :)

piggle

#2 HomeBrew

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Posted 23 August 2009 - 06:20 AM

I think you're right. Just make sure you use a long enough gel and run it long enough to achieve separation.

#3 jiajia1987

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Posted 23 August 2009 - 11:21 PM

you can make a very long gel at 1% or 0.8% and run it for 2 hours at 80V to run till the bands are separated out far away. There were times when I ran my gel for 3 hours to separate the two bands away.

#4 ivanbio

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Posted 24 August 2009 - 09:17 AM

Hi piggle,

Another thing you may want to consider, if your PCR is very clean (other than the primer dimers), is to run your PCR reaction through a PCR purification column, like Qiagen's QIAquick PCR purification columns. These columns work pretty much the same as their DNA purification columns. Anything smaller than 100 bp will pass through the column (your primers and primer dimers) and your 300 bp fragment will be retained in the column, which you can the elute and use directly for cloning. The advantage of this is that you do not have to deal with purifying your fragment from an agarose gel, which is known to cause cloning problems (agarose inhibits ligase). Another advantage is that you will very likely recover more of your PCR fragment using a column compared to extracting it from agarose.

Good luck

Ivan
Carlsbad, CA




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