Subcloning pcr products
myagi at u.washington.edu
Fri Oct 13 11:45:56 EST 1995
We had a lot of difficulty trying to clone PCR products with a Sal I site
at one end; I suspect that the enzyme doesn't like to cut near the ends
of DNA molecules (at least our tube never did). I assume you are adding
a few (4 or more) extra nucelotides at the end of your primer? Most RE
need at least 4 extra bases beyond the end of their recognition sequence
to cut efficiently. (Apologies if you knew this already).
We ended up using the TA cloning kit from Invitrogen to clone the PCR
products directly, then cutting out the inserts and recloning into the
vector we originally wanted to get into. More work, but in the end, more
productive than beating my head against the wall.
On 13 Oct 1995, Dr I.M. Clark wrote:
> We have produced an approx 500bp pcr product with a SalI site at
> one end and a BamHI site at the other. We have tried to cut and
> subclone this into a Sal/Bam cut vector with no success.
> Is there a particular problem with cutting such pcr products
> or using these particular enzymes?? Any hints or advice would
> be appreciated.
> Ian M. Clark
> Rheumatology Research Unit,
> Addenbrooke's Hospital,
> Cambridge. UK.
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