Explanation needed for DNA that "sticks" to wells

Hiranya Roychowdhury hroychow at NMSU.EDU
Tue May 9 14:30:06 EST 1995


On Mon, 8 May 1995, Richard Varhol wrote:

> 	I am using a semi-nested primer approach for the amplification of a 
> 	500 bp fragment.
> 
> 	I start off by amplifying a 1200 bp fragment in the initial PCR, and 
> 	then making a serial dilution of this, I amplify up a second time 
> 	with my internal primer.
> 
> 	What has been happening for a long time now, is that at times in 
> 	either stage of this dual amplification, there is a very large 
> 	distinct band sitting in the loading well, even after running 
> 	the loading buffer off the checking gell.
> 
> 	The controls are working fine, so I'm quite certain that the agarose 
> 	isn't faulty.
> 	
> 	Is there anyone, that can attest to this, or explain why this is 
> 	happening? I would greatly appreciate any insight on this 
> 	problem.
> 
> 	(By the way I have tried heat denaturing and I still have the DNA 
> 	brightly stained in the well)
> 
> 	Thanks in advance
> 	Richard
> 
> 
The problem stated is seen in preparations that contain either excess 
protein contamination or have proteins that have the propensity to 
bind/adhere to the DNA samples. These proteins are not easily extracted 
by conventional PCI extraction. Such problems, however, are rare in 
amplified samples since the amount of proteins (in the form of 
polymerases) should be miniscule. 
	I routinely clean such difficult DNA samples by using EthBr along
with PCI. The idea is to use about 0.5% conc. of EthBr which perhaps
inercalates and extends the grooves, allowing easier access for the the
PCI to "clean out" the DNA. One has to be cautious not to expose the
DNA-EtBr mixture to bright light (I keep the tube covered with foil).
Include amm.acetate to a final conc. of 2.5M (using a 7.5M stock). The 
EtBr/amm. acetate solution of DNA is then extracted with PCI as usual 
for one or two cycles. The remaider of the EtBr can be eliminated by 
extracting the final aqueous phase with buffer-saturated iso-butanol a 
few times prior to precipitating with ethanol. 
Hope this helps. Would like to know the result!

 

			>>>>>>>>>>>>>>>>>>>>>>>>>>>
			  Hiranya S. Roychowdhury
   			  Plant Genetic Engineering Lab.
			  Box 3GL, NM State Univ.
			  Las Cruces, NM 88003
			  Phone: (505) 646-5785
			  hroychow at nmsu.edu
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