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Purification of oligos for EMSA


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#1 Mindbomb

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Posted 29 December 2011 - 08:36 AM

Hi! I have 40 bp complementary oligos that I would like to label with biotin in the 3' end and then hybridize together. I wonder, what type of purification is necessary for subsequent EMSA reactions. How could I get rid of ssDNA? Could I detect the difference in some kind of gel and purify with a column? Thanks a lot for advice!

#2 Mindbomb

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Posted 30 December 2011 - 07:52 AM

some more questions - Surfing the internet I found some labs using PAGE purification of biotinylated oligos. Should I do this before or after oligo annealing? My oligos are at the moment only desalted.

Interestingly, I also found a protocol where the biotinylated and annealed oligos were purified from agarose gel using typical columns. May be this way I could get better yelds? However, would I be able to distinguish biotinylated/unbiotinylated and single stranded from double stranded DNA? After all, the oligos are rather short (mine range from 30 to 60, with average at 40bp). What type (%) of agarose and buffer and columns would you recommend!

Thanks again!




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