The efficiency test of the electrocompetent E. coli didn´t work. Probably the antibiotic concentration in the plates is wrong.
Taís
YeastGuard
New biobricks - New strategy
This evening we had a wonderful idea to try to solve our problem in screening the new biobricks in LB plates. While we wait the new primers to arrive, we decided to use pGEM T-easy vector as a way to add the EcoRI restriction site in the PCR products that already have XbaI and SpeI sites. This strategy will permit the part to be ligated to the biobrick vector avoiding unwanted ligations between the cohesive ends and consequently the hard screening we were performing.
For detailed information about pGEM T-easy vector system, see the Manual.
So today we started digesting both PCR products and pGEM vector using SpeI. We believe that this digestion will facilitate the correct insertion of XbaI end besides EcoRI site. After digesting, we did the ligation reactions, transformed and plated it in LB+AMP+X-gal medium. We hope that tomorrow we will find few blue colonies and a LOT of white ones! =]
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