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Purification of small (100bp) PCR products


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7 replies to this topic

#1 monsa

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Posted 11 March 2005 - 04:22 AM

I am trying to purify a 144bp PCR product. I will use this as a probe template so I want to make sure it is free of other PCR component (i.e., dNTP, taq, etc) and also get rid of an extra, faint band of higher (~300) molecular weight. I tried with the Qiaquick columns (Qiagen) but without success. According to the manual, they are good in the range 100bp - 10 kb, so this may be the problem.
Does anybody have a good purification protocol for small (~100bp) PCR products?
Regards,
Monsa

#2 fred_33

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Posted 11 March 2005 - 04:27 AM

hi
i usually purify my pcr products by separating in an agarose gel and using the qiagen pcr extraction kit.

You can either try the b-agarase of NEB.
for rev :
http://www.neb.com/n...roductM0392.asp

Edited by fred_33, 13 March 2005 - 03:25 AM.


#3 pcrman

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Posted 11 March 2005 - 09:21 AM

Yes, you first separate the band by running a 2% agarose gel, cut out the right band, then recover the DNA using Qiagen PCR purification kit.

#4 postdoc2130

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Posted 11 March 2005 - 01:48 PM

My lab routinely use GenCatch PCR purification and gel extraction kits (Epoch biolabs) to clone small fragments of 120-400 bp (DNA/RNA-binding motif) with very satisfactory and consistent results.
And their prices are cheaper too.

#5 methylnick

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Posted 12 March 2005 - 08:34 PM

I can suggest a cheaper alturnative. And you would be sure to not lose the product because it may not bind to the column.

You can run your PCR on a agarose gel, cut the band of interest out, place into an eppy and add 100uL of TE. Centrifuge to ensure the slice of gel is in TE, or you can now mash the gel up with a pipette tip. Then place the tube into a 60C heat block for about 15 minutes. Then spin the tube at maximum on a table top centrifuge for 20 minutes. Aspirate the supernatant which now contains only the DNA of interest. :angry:

Good luck!

Nick

Edited by methylnick, 12 March 2005 - 08:35 PM.


#6 fred_33

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Posted 13 March 2005 - 03:23 AM

hi
well methylnick, i have to do a pcr tomorrow, i will try your protocol!!!

#7 mario2004

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Posted 13 March 2005 - 09:19 AM

Hi Fred, please let us know your testing result.

Hi Nick, will there any problem for such extracted DNA in downstream applications such as ligation, sequencing...

Thanks you guys for sharing.

Mario

#8 methylnick

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Posted 13 March 2005 - 08:35 PM

Fred,

good luck with it!

Mario,

I have managed to ligate the product into a TA vector, sequence, do nested PCR and restriction digest it.

I think as long as the amount of supernatant used is minimal it should be fine.

Good luck with it!

Nick :angry:




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