Digesting SAS-4 and Amplification

Last week, my supervisor and I decided to digest our SAS-4 plasmid to go from circular to linear. This may improve the efficiency of the PCR to amplify the fragment for injection. We decided to digest the plasmid using PVUII, an enzyme that has only one site on our plasmid outside of the 16 HA tags. I proceeded with the suggested protocol to digest the plasmid, using a CutSmart buffer and incubated the mix for 37C for 30 mins. Despite the protocol suggesting a 15 minute incubation time, the enzyme/buffer available in the lab was expired, therefore to be safer I incubated the mix for extra time. Once the plasmid was digested, I purified the mix using the QIAGEN purification kit, using the same procedures as used previously when purifying my clones. I then did a temp grad PCR to determine what the optimal conditions would be for amplifiying the linear fragment. This ended up being 72C, presenting a largely clean band.

temperature gradient PCR for linear SAS-4

Based on these reuslts, I then repeated this PCR 4 times with an annealing time of 72C to ensure that enough SAS-4 was amplified for the injection. However, once again, when measuring the concentration of SAS-4, it was too low. Additionally, when I ran this amplification on a gel, the lane was not as clean as the temp gradient, presenting several additional bands.

As frustrating as this was, we were back to the drawing board, and had to decide what the best way forward for this. It is likely that we will use this mix and inject it into the worms despite not having a clean singular band, otherwise my supervisors have been discussing redesigning the insert/plasmid so that it easier to amplify.