enygmatic restriction by BamH1 - (Jan/18/2007 )
I have done a restriction on a plasmid by using the BamH1 endonuclease (recognition site GGA TCC)..
It seems (after sequencing) that the enzyme das recognized two restriction sites "GGA TCT" and "GGA TCC" !!!
Did anyone of you encountered such a thing ? is it possible for un enzyme to cut into a site different from its supposed restriction site ??
I'll be glad to hear your comments ...thanks in advance for your help
Youssef
well, if you have too much enzyme for a certain quantity of DNA and/or too much salts, enzyme may exibit star activity... or recognize some alternative site.
Have you done 2-3 rounds of sequencing to be sure?
You may face an other problem : a mutation dunring ligation step.
Have you done 2-3 rounds of sequencing to be sure?
You may face an other problem : a mutation dunring ligation step.
Fred,
Thanks for your answer. Indeed, I used a little excess of the enzyme to digest the plasmid ,, that may be a good explanation of what happened ,, Otherwise the sequencing was done by a special company of BioMol and I don't know how do they verify the sequences.
Merci pour l'intérêt que vous avez porté à ma requête
Olepaul0
you can look at the chromas trace. The sequence itself is generated by a programmed that reads this trace. Occationally the machine makes a mistake. So if you look at the trace, you will be able to determine if the reading was acurate or not.
Another way to verify your sequence data (especially in regions some distance for your primer), would be to sequence your fragment from both the forward and reverse direction. If the two sequence reads agree, the your answer.
And as mentioned by fred_33, you should sequence 2 - 3 different isolates of the same clone, just in case that there is a ligation error. (Sometimes the ligase doesn't stick your DNA fragment correctly and misses out a base)
Another way to verify your sequence data (especially in regions some distance for your primer), would be to sequence your fragment from both the forward and reverse direction. If the two sequence reads agree, the your answer.
And as mentioned by fred_33, you should sequence 2 - 3 different isolates of the same clone, just in case that there is a ligation error. (Sometimes the ligase doesn't stick your DNA fragment correctly and misses out a base)
Thanks Perneseblue
The sequencing was done by one primer (so one direction..),,
I just want to make it more clear; it is not just a problem of one nucleatide mutation.... in fact as it seems that the enzyme has cut into site different from its recognition site so there is "in addition" a deletion of about 30 nucleotides within the sequence (and this was confirmed by observing chromas,, so there is no doubt about that).
thanks for your help guys ,, that did really help
regards
Youssef
Be sure to use the supplied buffer for BamHI. Although it is active in a wide variety of buffers, NEB supplies a specific buffer for BamHI designed to reduce star activity. Also, of course, use the correct number of units.
I use EcoRI, which has the recognition site GAATTC, quite similar to BamHI. Although I can't be certain I often suspect star activity with this enzyme. Maybe this recognition site and sites like it are prone to star activity, because they are chemically similar. Just a guess
Thanks for your help guys..
how long did u digest ur plasmid with the bamHI??? BamHI show star activity if u incubate for too long time