I have fluorescent labelled DNA oligos, having a single BamH1 site. I'm screening some compounds which may affect the enzyme cleavage efficiency. My problem is that the oligo is not getting cleaved at all even in the absence of my compound. There are 5 bases upstream to the cleavage site. I have varied oligo conc., enzyme quantity, and time. I would appreciate all leads in solving my problem. Thanks in advance..
whether the buffer is compatible with the enzyme? Please share the sequence of oligos with RE site and gel images of RE digestion if possible for troubleshooting of this problem.
I'm using NEB 4 buffer, the enzyme is Hi-Fidelity version. The sequence of the Oligo is 5' CCATAGGATCCTTAACCTGG. Its labelled at 5' end with Alexa 488. I don't have any gel images for the digestion but I cannot see the digestion happening on the gel. I'm using a 2.5% agarose gel with a 10bp DNA Ladder.
Restriction Enzymes are used on double stranded DNA. Are you using linker or just oligo?
Better to use a longer PCR product for study.
dear Mary
your RE might be invalid for many reasons like improper storage conditions or erratic buffer concentration (usually 1X), or your DNA sample had insufficient amount or degraded
please try to verify your experiment conditions
best regards
Thank you all for suggestions. Jaspreet I'm trying PAGE today..Vasudha I annealed my oligo and checked the annealing also. So its double-stranded now. Meelad I have used controls to check activity of RE and confirmed it is working..Amount of DNA in reaction is 50 ng.
Thanks
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