Restriction enzyme sites mutate after ligation? - (Oct/18/2007 )
Dose the mutation of restriction enzymes site after ligation happen frequently??
I use two enzymes, AgeI and AseI, to cut my vector and insert DNA, then make them ligate together by sticky-end ligation.
After ligaiton, I sequenced the insert DNA which had been confirmed by few restriction enzymes.
The majority of the sequence result are correct, however, the most important part(my original restriction enzyme site, AgeI and AseI, used to sticky-end ligation and following-up cloning) just "disappear"!!! ( for example: original site ATTAAT is replaced with ACGACC)
HOW COME and WHEN dose these mutation happen?? If the restriction site have mutated, how can I cut the insert and vector and ligate them together???? It is really weird and so so unbelivable...
Is there anyone helping me to explain this situation??
I havnt seen when sites are mutated. Check the sequencing again, I mean look at the raw data to verify that sequence has changed.
how many isolates of your plasmid do you have?
Do check again.
But mutations at the ligation point do occasionally occur, it is not often but I have seen a few examples myself. I don't actually know the answer and can only speculate.
Since this type of mutations are rare, picking another isolate of your plasmid should produce a normal plasmid.