restriction enzyme question!!

[mk_2007]

Hi! Another question!! If you clone a gene, lets call it B, as a Xho I/Hind III fragment into a vector, then digest this vector with Hind III/Sal I, why would Xho I and Sal I sites be missing from the map?

I know this is because, there is no restriction site for the Xho I and Sal I enzymes in the vector, but why? what could have happened to delete a restriction site?

 

Help would be much appreciated!!!

[ecoli]

I would need more information to answer this, I think. Show us the genetic map, what you did, and what the problem is.

 

Are the restriction enzymes XhoI and SalI no longer working after you insert the gene? Have you made sure that they're compatible enzymes? (in terms of the buffers you can use).

[Mr Skeptic]

Another thing that can happen is that the site that two restriction enzymes act on can overlap. Then if you splice it into a vector with different base pairs near the restriction site, it might not recreate the site for the second restriction enzyme.

 

Eg if you have restriction enzymes for A'AGCTT and CTT'AAG, then you can cut the strand CTTAAGCTT between the A's, then stick it to any strand ending in A, but it doesn't need to end with CTTA, so your second restriction enzyme won't work.

Edited October 29, 2008 by Mr Skeptic

[CharonY]

Yeah that is basically the answer, I think, though the OP is misleading. I think this is homework and

I assume what he/she meant was that the insert was digested HindIII/XhoI and the vector was digested HindIII/SalI.

It was not, as the post implied, first cloned and then tested by double digestion, as the latter would probably not make much sense.

The important bit here is that SalI and XhoI have different recognition sites, but produce compatible ends (as In the scenario outlined by Sceptic). And I assume the confusion in the OP was derived from the fact that the "clone" was used erroneously here.

 

(Actually I have used similar questions in exams before)