nonspecific band after purification - (Aug/11/2005 )
After RT-PCR I got a desired band of size 800bp. After phenol purification, ethanol precipitation and washing with 70% alcohol I again checked on gel but this time instead of getting a single band I got another band also of size 500bp and which was more intense than 800bp.
What might be the reason.Should I discard the sample.
Any suggestion will be highly appreciated.
This has happen to me too. What that 500 bp band is, is reversibly denatured 800 bp band. You can help it renature by putting it into a 37C water bath for a couple of hours-overnight. That will help some of it renature.
You can avoid the problem by using Quiagen PCR purification kit.
Small DNA fragments can denature easily if there is no salt present. I suggest adding some NaCL (~150mM) and renaturing (heat up and let cool slowly).
Improved automated DNA sequencing traces