how to remove biotin from DNA - (Apr/25/2007 )
Hello
Does anyone know how to remove the biotin from 5 prime of double-stranded DNA. I am trying to do T-A clone for sequencing. the primers were biotinylated in PCR, so I can not clone the fragments from PCR.
THanks
Walter
Is this the standard biotin adduct?
There are variations where the carbon tail connecting the biotin molecule to the DNA strand has photo-cleavable molecules inserted. Thus UV exposure will release the DNA, perfectly clean.
However if you biotin comes on the standard adduct, I believe your DNA molecule is very much stuck to the biotin. Anything that will break a phosphoether bond will certainly break the more unstable phophoester bond of the DNA backbone
Was a unique restriction site engineered close to the 5’ end? Is PCRing your DNA possible?
There are variations where the carbon tail connecting the biotin molecule to the DNA strand has photo-cleavable molecules inserted. Thus UV exposure will release the DNA, perfectly clean.
However if you biotin comes on the standard adduct, I believe your DNA molecule is very much stuck to the biotin. Anything that will break a phosphoether bond will certainly break the more unstable phophoester bond of the DNA backbone
Was a unique restriction site engineered close to the 5’ end? Is PCRing your DNA possible?
Thank you for you help. It is standard adduct of biotin. the primers worked well and I already got my target gragments. There are not any restriction sites at 5', and I just want to clone them by T-A vector for sequencing. How can I?
Thanks again.
Walter
Since you have the obtained the biotinylated PCR product, is it possible to obtain/purchase a set of primers without the biotin moiety? Using those primers you could reamplify your product, using teh biotinylated molecule as the template.
Other then that, I don't see anyway out.