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Hi Amit, Normally, we optimize annealing conditions with labeled DNA (cy5, FAM) - adding MgCl2 and KCl usually improves annealing efficiency. You can detect fractions of single and double stranded DNA on standard PAGE gels. We then use the same condition to anneal the crystallization reagents. At times we order double stranded DNA cut from PAGE gels by the supplier - but that's expensive. Also, you can purify protein-DNA complexes using gel filtration - your protein shouldn't co-elute with ssDNA. There are also dyes that specifically stain double stranded DNA but we haven't tried those. -ralf ------------------------------------------- This email is confidential and may be privileged. If you are not the intended recipient, please delete it and notify us immediately. Please do not copy or use it for any purpose, disclose its content to any other person. Thank you. ------------------------------------------- -----Original Message----- From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of amit sharma Sent: Friday, December 18, 2009 2:53 AM To: [log in to unmask] Subject: [ccp4bb] DNA prep for protein-DNA complex crystallization Dear CCP4BBers, Sorry for the non-CCP4 question. I intend to set up some trials for protein-DNA complexes. In this regard, I have a few queries: 1. After annealing the oligos, how do I ensure that what goes into trials are only the annealed dsDNA and not other forms such as ones carrying hairpin loops. 2. Should I visualize it on the agarose gel and then try to get the dsDNA band? Sorry for these queries as I have little idea about this. Many thanks in advance. Cheers -- Amit Sharma, Ph.D. Postdoctoral Fellow, Department of Biophysics, Johns Hopkins University, Baltimore, MD21218