Plasmid Amplification with PCR

After the recieving plasmids from sequencing and confirming which plasmids actually contained the construct, I was able to select the correct samples and begin amplifying them using PCR. This process will ensure a sufficient amount of plasmid is available to be later injected into the C. elegans worms.

However, I did face a few difficulties! Unlike typical PCR which utilises Taq polymerase, the enzyme I was using was Phusion. Given a new enzyme I had to design the PCR experiment and chose the following design:

Initial PCR protocol designed using Phusion protein standard protocol. Annealing temperatures depend on which gene was amplified, SAS-4, SAS-6 or SPD-2 respectively.

However, results from this PCR demonstrated no amplification when compared to just plasmid. My Post-doc supervisor and I realised the ineffective PCR was likely due to the high concentration of template DNA in the sample, where I had used 250 ng, where ideally I should have used 1pg-10ng - whoops!

This PCR was then rerun using the correct concentration of template DNA (started with 5ng) and decided to run a gradient PCR. This would allow me to identify what the optimal annealing temperature would be for future PCR runs. This was done as a range from 50C to 63C and amplified DNA was run on a gel to show which annealing temperature is best fit for the PCR. Unfortunately, results from this PCR demonstrated a second band - possible contamination? Again, another gradient PCR was run, this time with a higher annealing temperature range from 63C to 72C to see if this had any changes on the amplification.

Gradient PCR set up for all three genes, SAS-4, SAS-6 and SPD-2

Results from this new gradient PCR suggests an optimal temperature for each plasmid, however a second (faint) band can still be observed. This indicates that the PCR needs to be further optimised to ensure there is no second band and done at the correc

annealing temperature.

SAS-4 PCR results

SAS-6 PCR results

SPD-2 PCR results