problems with gel mobility shift assay

Zhang Zhiying zhangz at ERE.UMontreal.CA
Tue Aug 20 13:26:35 EST 1996


I have problems with gel mobility shift assay. The DNA probe I used is 
partial of a fish retrovirus U3 sequence. I first pcr the fragment (about 
200 bp) with two primers which suppose of 5'-OH and the PCR product was 
purified from agarose gel, end-labelled with gamma-32P ATP by T4 kinase, 
purified with G50 spun column. The labelled fragment was used in nuclear 
protein  binding reaction. I run the binding reations and the control 
without protein on both 6% and 3% nondenaturing acrylamide gel at 200V 
for 3 to 4 hours. The problem is a strong band on the top of the gel 
followed by several pale bands on film  but no band shown up for the 
control. I just wonder Where is the free probe band (control)? Could 
someone help me out. Thanks in advance.  



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