I am trying to label a probe with flurescein dye through nick translation. In the previous step, PCR with the respective primers is yeilding a desired amplicon of 250 bp on agarose gel. But after the procedure of nick translation and labelling, i am getting a very small fragment of less than 50 bp and not the size (150bp) as desired. Please give your suggestions
Nicking is random process and the fragments will always be smaller than the input DNA fragment size. Try titering the DNAase (stepwise reduction of activity) as you likely are introducing more than one nick per template.
Try decreasing the length of time the nick translation reaction runs for. The longer it goes on for the more the DNA gets broken up.
Thanking you all for the suggestions. I have tried to reduce the time from 2 hours to 1 hour 20 minutes and 1 hour 40 minutes but still getting a very small fragment of less than 50 bp, please advice
250 bp is a small amplicon, try Random Primer Labeling it may provide better results. Alternatively, for Nick Translation decrease DNAse concentration.
For the size of your fragment nick translation is not a good choice, its very small. For this size of fragment it is better to use PCR with labeled nucleotides (biotin or digoxigenine)
250 bp is a very small amplicon for Nick translation. Even for a probe as well.
You could try to use a PCR labeling procedure, or clone your amplicon in a plasmid and then the Nick translation. I think that is the better choice due to the small size of your probe.
It doesn't matter the labeled nucleotide you use.
Good luck
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