Fri, Sep 03 21

VR transformations and VR fragment isolation #

Gibson assembly product transformation #

Previously I had completed Gibson assembly reactions for inserts 1-16 but had not yet transformed cells and kept these samples in the freezer. Since then I learned about the homology between the inserts and the vector Thermo used to synthesize inserts into which caused PCR products from vectors to be larger than those from fragments. Since samples from this round of Gibson reactions had not been digested with BglII prior to the reaction I did not use reactions with inserts that were contained in fragments.

I transformed inserts 1, 2, 4, 5, 7, 10, 11, 13 and 14 into Invitrogen chemically competent cells following the supplied protocol. I also used the provided pUC plasmid as the positive control and Gibson isothermal mastermix as the negative control. After transformation and recovery I plated all samples onto Agar + Amp plates and placed into the 37C room to grow overnight.

VR 29, 30, 21 PCR and GblII digestion #

Following up on the issues with PCR amplification of these fragments using Phusion polymerase, I reran the PCR reaction for these inserts with lab Taq polymerase and then diluted VR-31 to 50ul and digested with BglII overnight at room temperature as I was leaving the lab by the time the PCR was finishing. Will extract these fragments tomorrow when I come in to retrieve the transformed colonies.