Re-attempts of SAS-4 and SAS-6 amplification

Given the several failed amplifications of SAS-4 and SAS-6, my supervisor decided that it would be best to proceed with the injections even though running gels of these PCRs demonstrates the presence of another band (otherwise there is more than just the insert that gets amplified). We resulted using the PCR conditions for SAS-4 to be 71.4C and 3% DMSO, and for SAS-6 to be 67.4C and 5% DMSO.

Following amplification and purification, the DNA concentration was measured to identify how much DNA would be injected into our worms, and was done using the nanodrop. At this point, the past few stages were becoming easier for me given the several repeats of PCR and purification that needed to be done. Furthermore, I had already had some experience using the nanodrop during my third year undergraduate project, so I was familiar when using it. When recording the concentration of my DNA, the nanodrop readings demonstrated a very low concentration (33.85 ng/ul and 39.95 ng/ul respectively). With such a low DNA concentration, we could not proceed with creating the injection mixes/injecting worms as not enough of the insert was available, hence we were faced with another challenge.

In the end, I repeated the PCR for both SAS-4 and SAS-6, this time using 35 PCR tubes filled with 50ul of PCR product (where previously I had done 20 PCR tubes with 20ul PCR product). This was to ensure that there was enough DNA being amplified for the injection mixes. Additionally, where I previously did 30 PCR cyles, this final time I did 35 cycles to be extra sure that enough PCR product was being made. Although this many PCRs were required, by the end I was extremely worn out from pipetting into several PCR tubes!

After amplification, the DNA was pooled with the previous PCR product and purified using the same purification techniques - QIAGEN PCR purification kit as well as AMPURE beads. This round, PCR product had a concentration of 190.4 ng/ul (SAS-4) and 169.95 ng/ul (SAS-6), a huge improvement from the last PCR. These samples were then ran on a gel to identify how clean the PCR product was.

Unfortunately, we were not satisfied with the gel for SAS-4, resulting in several extra bands. However, SAS-6 did appear clean enough, with only one extra band. Despite not being fully clean, every PCR resulted in the presence of this second band, therefore we decided to continue and prepare an injection mix for SAS-6.

SAS-4 however needs to be revisted again to ensure the correct insert is amplified enoguh for injections. This again was quite frustrating but through this project I am identifying that several failed attempts is normal in science and given that this PCR has been repeated several times, all with failed results, is likely due to a contaminated reagent. My PI went through this with me and identified that although we could go repeat the PCR changing one reagent at a time, it is better to start with everything fresh - a new dNTP aliquot, primer aliuot, new reagents, etc. This (with my PI's experience) is likely to result in positive results, and less time would be used up trying to identify which reagent has been contaminated/gone bad.