PCR optimization

My PCR primers work! But the PCR protocol itself is not optimized to these primers since we saw laddering. Carolyn suggested I finagle with the thermocycler settings to try and eliminate the laddering

2024-05-31

Carolyn showed me how to set up a gradient on the thermocycler to test multiple different temperatures at once.

Figure 1. Thermocycler gradient settings

I then ran a PCR using DNA from sample 172 (and a bit of 47 since I ran out of 172) on the gradient to see if there was a better temperature that would lead to less laddering.

Figure 2. Gel image for gradient gel. 64ºC is the first lane and 54ºC is the last lane

We’re still getting laddering. There is less laddering at 64ºC, but the band is also not as strong.

2024-06-03

It’s possible that the laddering is occurring because the PCR protocol has a 90 second extension time. Maybe by having a slightly shorter extension time of 60 seconds instead, I can reduce the laddering! I used the same gradient as last time, but changed the extension time to 60 seconds.

Figure 3. Gel image for gradient gel with 60 second extension time. 64ºC is the first lane and 54ºC is the last lane

The shorted extension time does seem to reduce laddering a touch! When I showed the gel to Carolyn, she suggested I move forward with the 60ºC + 60 seconds on the thermocycler and test out the Alul restriction enzyme I’m borrowing from ToxLabs. Based on that, we can determine if we need to optimize the PCR more.

Going forward

1. Set up cold room
2. Obtain MA crabs!
3. Tailor PCR protocol for new primers
4. Develop restriction band digest for genotyping
5. Develop heart rate protocol