[Q] How to purify small PCR frg. ?

Sailesh Surapureddi SaiSu at MCB.LiU.SE
Wed Jul 19 04:14:10 EST 1995


>To: methods-and-reagents at net.bio.net
>From: p_rudolph at morph.spacenet.de (Peter Rudolph)
>Subject: [Q] How to purify small PCR frg. ?
>Date: 19 Jul 1995 08:50:20 +0100
>
>Hi netters,
>
>recently a coworker of mine ran into a problem:
>She created a library with PCR using randomized primers,
>then she digested the fragment and want to ligate it into a vector.
>The complete PCR fragment is 96bp, after digestion she gets
>two fragments: 29 and 67bp. She separated them by gel
>electrophoresis on a 2.5% AG.
>The problem is now: How to purify the bigger fragment from the
>gel slice with a really *high* yield?
>She tried freeze-and-squeeze and Qiagen-gel-ex but got
>only very low yields.
>Any suggestions?
>
>Any help will be highly appreciated!

Hi,
	With qiagen I have noticed, to get a better yeild, you have to add some 3M sod. 
acetate, they dont subscribe it in the bold but mention it as a footnote in the 
second step, i,e after the addition of glass beads and warming. 
	The amount of DNA binding to the glass bead will also improve if you warm the 
total mix to 65 C instead of 50 C. 
	Ofcourse the last step i.e drying should be for 30min and elute your DNA twice. 
Besure to add a little more of glass beads then prescribed.
Hope it works for you now, all the best.
Sailesh.



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