PCR ASAP

Well, as I feared, the "no grind" extraction was fruitless. The "wide bore" extraction was unfortunately not a resounding success either. There was DNA, but it appeared to be sheared (again). Another interesting point to note, is the distance the samples traveled through the gel. As far as my understanding goes (and from many, many conversations with people more in the know than I), genomic DNA typically does not travel as far as it did in my gels. Genomic DNA is relatively large and therefore should not migrate very far in the gel at all. Could the additional observed travel be a result of shearing making the molecules smaller and therefore easier for them to travel in the gel?

As the time crunch is officially upon us, I have decided to see if, within the potentially sheared DNA samples, there is something the chloroplast primers can identify and reproduce. It is sort of a Hail Mary at the end of the semester in order to see if I can get some form of PCR results to use in comparison of the individual subjects.

For PCR, I mixed 550µL of master mix with 11µL of chloroplast primer to create a stock solution. 20µL of this stock solution was added to each PCR tube. Then, I added a combination of DNA extraction sample and Ultra Pure H₂O totaling 20µL. For each of the three DNA extractions I conducted 3 PCR reactions: tube one held 1µL of sample, and 19µL of Ultra Pure H₂O; tube 2, 2µL of sample, and 19µL of Ultra Pure H₂O; tube 3, 3µL sample, and 17µL of Ultra Pure H₂O. The protocol or pattern for the PCR cycle is as follows:

1. Initial Denaturation: 94°C for 2 minutes
2. Denaturation: 94°C for 1 minute
3. Annealing: 59°C for 1 minute
4. Extension: 72°C for 2 minute
5. Steps 2-4 repeated 40 times
6. Final Extension: 72°C for 10 minutes
7. Hold: 4°C indefinitely

Tomorrow I will cross my fingers and run another gel with the PCR results. Here's for hoping!

Preparing the samples for PCR.