Project:Sex typing with amelogenin/results-20120215
Bugs-based course re-alignment
This week Bugs appeared, as mysteriously as he had disappeared previously, and had a look over our work. He pointed out a bunch of things we could look at:
- We should use distilled water.
- We are using too much primer. We were using 100µM concentration, with 10µL in the PCR reaction vessel. Bugs tends to add 3µL of (100 µM conc) primer to 97µL of water, and then use 1 or 2 µL of that in reaction. Too much primer probably shouldn't prevent the reaction from happening, but it could definitely make things messier.
- We were using the wrong concentrations in the reaction. The master mix is designed to work with specific concentrations of primer and template, and if those are messed up the entire thing won't work.
- 25µL master mix
- 2µL F primer
- 2µL R primer
- 10µL DNA
- 11µL water
… for a total volume of 50 µL. We could also halve all these amounts, though that of course will result in less DNA.
Bugs was a bit bewildered by the primers themselves (as we had been, for the same reasons, when we did our first PCR run): the F primer has a melting temperature of between 60 and 72 C (depending on which source you consult), and the R primer has a melting temperature of between 47 and 56 C. Usually primers have melting temperatures within a few C of each other, because the annealing phase is then set to be a couple of degrees below the primer melt temperature. However, one paper (see the last section of results from the 12th) has the annealing temperature set at 65 C. We don't know what the correct option is, so we will just have to try a few.
I tried two new DNA extraction protocols. The problem with both of these protocols is that we have no idea how much DNA we are actually extracting. Perhaps we are extracting too much. "There is really no lower limit," says Bugs, because PCR will amplify the DNA.
- 500ul of chelex solution 5%.
- Cheek scraping using pipette tip. Pipette up and down in chelex solution to mix.
- Incubate for 10 minutes at 56 C.
- Incubate for 6 minutes at 98 C.
- Vortex samples (manually :/)
- Spin tube for 5 minutes in centrifuge.
- Take 50ul supernatant.
- Spit into a PCR tube. Add a few µL of water if necessary (to take it up to a few hundred µL, or about halfway up the PCR tube)
- Cheek scrapings into tube, pipette up and down
- Add a few grains meat tenderiser and mix.
- Leave for a few minutes.
- Add rubbing alcohol and mix.
- DNA precipitates out of solution and appears at the interface of alcohol and saliva.
- Extract the DNA (pipette) into a fresh PCR tube. Leave it to "dry" -- wait for the alcohol to evaporate.