PCR reamplification problem

H. Allen Sylvester asylvest at ASRR.ARSUSDA.GOV
Fri Jun 20 14:19:30 EST 1997


Ed,
	I have recently had success reamplifying PCR products (to 
eliminate low molecular weight smears) of about 500 bp.
	I run the PCR product in a TAE agarose gel to verify what I 
have.  I then put the gel on aluminum foil on the UV light box to block 
UV from all lanes except the one I am working on and only turn on the UV 
for a few seconds when I need it.  I take a Pasteur pipette and stab it 
into the gel in the band of interest. I rock the pipette to break the 
vacuum as I remove it, otherwise the gel plug usually stays in the gel.  
I then carefully blow the plug out with the pipettor, into a 0.5 ml vial 
and add 25 ul of water.  I let it sit at room temperature overnight for 
the DNA to diffuse and vortex it before I use it.  For 1700 bp you might 
want to crush the plug somewhat.  I never had much luck with freeze-thaw.
	I then use 0.5 to 2 ul in a 10 ul PCR reamplification, depending 
on how intense the original band was and how well a specific PCR reaction 
works.  Generally this eliminates the extraneous trash and gives a more 
intense band.  Sometimes I have to repeat this because trailing smaller 
molecular weight trash seem to come along the first time.
	This is my modification of suggestions in several earlier posts 
over the last few months.  Disposable plastic pipette tips would probably 
work about as well, except if the plug moves up away from the opening, it 
is a hassle to get it out.  Similarly, when I tried to suck the plug up 
into the pipette, it went too far and would not come out without breaking 
the pipette.

Allen
H. Allen Sylvester
USDA, ARS, Honey Bee Lab, Baton Rouge, LA



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