SalI and double digestion. - (Jan/22/2013 )

Dear members,

I have performed restriction digestion with SalI and SmaI in a sequential mode. While cutting with SalI itself the vector and the insert got separated. (when it can only happen with double digestion). So, to check it further, when I purified the gel and cut the insert and further digested with SmaI, I can see a faint band of my gene only with the correct band size as per the case of SalI. This apparently seems to be correct as SmaI only has the gene to be excised, no vectors to separate my gene from. But, my query is why the case happened like this with SalI? why did it separate my gene of interest at the single cut? is there any specific property for the same? or I am missing something on the way.. Please if anyone can guide me...

Note: SalI and SmaI are the enzymes that do not cut the insert. So, I selected the same.

Keenly looking for some guidance at the earliest...

Thanks in advance..

I dont really understand what you are asking.

You have a plasmid with an insert, right?
THen you used SalI to cut the insert out of the plasmid.

Thn you ran a gel to seperate the insert from the vector.
You then cut the insert of a certain size out of the gel and incubate this with SmaI?

In the end, after rhe SmaI digest, you just have the same size of insert as you had after the first digestion with SalI.

IS this your question?

If so: why wouldnt it be normal that the size is the same? If SmaI can not cut in the insert, as you said (This apparently seems to be correct as SmaI only has the gene to be excised) then its normal it stays the same size...

Or did you cut both a new sample of plasmid + insert with the SmaI ? (meaning you did the same for SalI and SmaI => you started with an intact vector+insert?)

So, I recently contacted New England Biolabs about issues that I've been having with SalI and one of the things that they mentioned was the "star activity" of SalI.  Essentially what this means is that SalI is not a very specific cutter, so if there is a sequence that is close to the cut site for SalI, it will cut that site as well.  For instance, if SalI recognizes a sequence of 6 bases, it might also cut a different site where there are only 4 bases instead of the full 6.  What this means is that SalI can cause overdigestion.  Not a good thing really, but supposedly with their HF version of their restriction enzymes, this issue is resolved.  I haven't tried the HF versions, but I am considering it as they don't cost any more than the original.