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Restriction digestion problems

cloning

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3 replies to this topic

#1 biochemical mayhem

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Posted 09 June 2012 - 03:31 PM

hi dear friends, I have one vector having restriction sites 3 bases differences into which i want to clone my insert having the same restriction sites. I have no other options to pur other restriction sites. But i could never confirmed if my vector is cut by both the enzymes. Can somebody help me figure out how we can be sure that our vector is cut with two restriction enzymes that have sites 3 bases apart?
This will help me a lot...Thanks in advance.

#2 Kamran

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Posted 10 June 2012 - 03:43 AM

If both enzymes generate non-compatible ends then linearised vector would not be able to self-ligate. However, if one or the other enzyme is not working then the linearised vector would self-ligate very efficiently.

Try to do self-ligation assay.

#3 phage434

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Posted 11 June 2012 - 04:26 AM

I think what you are saying is that your RE sites are three bases apart. This may or may not be sufficient to cut at both sites, depending on the enzymes. Some enzymes will not cut near the end of linear DNA fragments.

When you say you have no choice of enzymes, this is not true. You can always use PCR to amplify your vector (not the insert) to create whatever sites you want by putting 5' RE sites on the oligos. Remember to put a 5' spacer region after the RE site to solve the problem of end-cutting. Of course, you must avoid other cut sites for the same enzyme in the vector.

#4 mkb323

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Posted 11 June 2012 - 03:56 PM

you can digest your vector by enzymes separately : 1st enzyme digest well .for 2nd enzyme ,digest effciency will decrease because of 3 base fo anchor ( for 2nd digest add more enzyme and increase incubation time ) .
what RE you use for digest ?





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