I created 5’ deletion constructs of a fragment of interest by PCR. The template is the entire fragment of interest subcloned into the MCS of pGL2Basic (Promega) between the SacI and BglII sites. Forward primers are complementary to the fragment of interest with a SacI site linked on the 5’ end with a preceding 3 base cap. Reverse primer for all amplicons is complementary to the part of the plasmid downstream to the MCS, and has a BglII site on the 5’end with 3 base cap. The strategy is to digest the amplicons with SacI then BglII (ethanol precipitation in between, as the two restriction enzymes require mutually incompatible buffers), separate on gel, extract, and ligate into a vector prepared by digesting the template in a similar way, finally transforming DH5alpha E Coli cells.
We have changed the gel extraction kit (Quiagen silica gel filter to Genomed Jetsorb), changed TAE brand for gel.
Because of concern that the SacI site was not being cut, as only 3 bp separate it from end of DNA strand, we AT cloned the PCR amplicons into pGEMtT-Easy (Promega), obtained plentiful clones, digested with both enzymes and verified that the inserts were properly cut out by running the gel, and religated into the pGL2Basic derived vector. Still no go.
We have tried a different brand of T4 ligase. Worst of all, the positive control plasmid worked in the initial attempt, suggesting no problem with competent cells, ligase or lab problems (“bad water” or such). It seems the problem is in the DNA.
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