Hey everyone,
I'm trying to synthesize some DNA probes from genomic DNA, with biotin incorporated so that they can be used to blot northerns and visualized by methods of chemiluminescence. I'm unable to find a good method for PCR with biotin incorporation, can anyone link me any good protocols?
Thank you so much in advance!
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#2
laurequillo
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Posted 30 June 2010 - 07:39 PM
Michelle0505, on Jul 1 2010, 04:56 AM, said:
Hey everyone,
I'm trying to synthesize some DNA probes from genomic DNA, with biotin incorporated so that they can be used to blot northerns and visualized by methods of chemiluminescence. I'm unable to find a good method for PCR with biotin incorporation, can anyone link me any good protocols?
Thank you so much in advance!
I'm trying to synthesize some DNA probes from genomic DNA, with biotin incorporated so that they can be used to blot northerns and visualized by methods of chemiluminescence. I'm unable to find a good method for PCR with biotin incorporation, can anyone link me any good protocols?
Thank you so much in advance!
I used to buy the primers already with biotin, they were not expensive
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#3
phage434
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Posted 01 July 2010 - 06:03 AM
Two options: biotin labeled primer (easily ordered) and spiking the PCR reaction with small amounts of biotin-dUTP, which will be incorporated during the PCR reaction, replacing dTTP. You need to use a Taq PCR enzyme, since the proofreading ones will not add dUTP. Many companies sell biotin-dUTP. You might consider using DIG as a label instead of biotin, since it has a bit lower background. Roche has patents on DIG labeling, so all of the reagents are sold there.
