e-anderson at ski.mskcc.org (Eric C. Anderson) wrote:
>In article <47agm2$6qc at hippo.shef.ac.uk>, M.C.Behrendt at sheffield.ac.uk wrote:
>>> Please could any netters in the know tell me if ligation of PCR products
>> into a T-vector requires any special conditions and if these alter from
>> a normal ligation?
>>>the only special condition is that you don't need to gel purify the
>product first. just take an aliquot (with the TA vector from Invitrogen i
>always use 2ul of a 25-50ul reaction) of the product, mix it with the
>vector, buffer and ligase and go for it.
>We have a stricter rule in my lab. We don't "go for it" unless the PCR reaction contains a fairly bright band, and preferably a single dominant band, rather free of primer-dimers. It only takes a few hr to try PCR again, so if we don't have a nice bright dominant band, we do another PCR. (It takes days to screen colonies by PCR or minipreps or restriction digestion or sequencing...then to find out that the inserts are no good!) If we do have a bright dominant band, we then use 1 µl or
less of the PCR rxn, plus 6 µl of water from the Invitrogen kit, and
follow all of their directions (except sometimes, we use a different
host competent cell than provided in the kit). The kit is well worth
the money saved in personnel. (I don't work for Invitrogen or hold