Project:Sex typing with amelogenin/results-20120311
Results
Two separate DNA extraction protocols: Chelex and meat tenderiser.
Chelex: rinse mouth with salt. Spit small amount of liquid into tube. Add 500ul Chelex. Incubate at 98 degrees for 6 minutes. Centrifuge for 5 minutes. Take 10ul supernatant.
Meat tenderiser: rinse mouth with salt. Spit small amount of liquid into tube. Add equal amount of detergent. Vortex for 30 seconds. Add a few grains meat tenderiser. Vortex for 30 seconds. Allow to sit for several seconds. Add a small amount of rubbing alcohol. Observe DNA strands precipitating out of solution. Take 10ul of liquid and DNA strands. Incubate at 70 degrees for several minutes. Add 5 ul water.
PCR was set at D=94 degrees, 1 minute; A=57 degrees, 1 minute; E = 72 degrees, 1 minute. I used 5 ul of sample, 6 ul of primers, 12.5 ul of Taq master mix.
Result was visualised on 1% agarose gel stained with 2ul/40ml of ethidium bromide under 300nm UV. DNA ladder was visible. Both samples showed a faint blur running ahead of the ladder and no other information.
I conclude that the DNA extraction technique is inadequate.
Things which could go wrong
1. DNA extraction and isolation
1a. Not extracting enough DNA
We are supplying a tiny sample.
Resolutions:
- . Try a larger sample: use a test tube rather than a PCR tube.
- . Try to extract DNA from another source. This could get gruesome: likely sources are other bodily fluids (blood, stool, semen -- so, yeah, let's try to stick with buccal cells); or flesh.
- . Be more rigorous about extracting DNA: use a hook rather than a pipette (though actually the pipette seemed fine)
- . Investigate more DNA-extraction methods
1b. DNA is contaminated by proteins, or by other things which inhibit PCR.
The most plausible contaminants include:
- Protein (from cells)
- Alcohol (from the DNA extraction method)
Resolution: use Chelex to bind proteins. Use triton X rather than regular detergent. Ensure that the alcohol has dried (this requires more research). Use water to re-hydrate the DNA.
2. PCR
2a. Chemicals are past their use-by date.
We are using old master mix. We could try to order more master mix. We have tried replacing the master mix previously, and didn't see an improvement -- but this is not conclusive as we have refined our process since then.
Resolution: Order more master mix. (NFD: ordered on March 11 2012)
2b. Wrong reaction volume.
The master mix documentation includes a recommended reaction volume. We were not following this initially. We are now following it.
Resolution: No action.
2c. Wrong DAE lengths.
Amelogenin primers show up a lot in the literature. No two papers use the same DAE lengths.
Resolution: Try several likely DAE lengths. D=60s, A=60s to 120s, E=60s to 180s seems to be about the range. In general it can't hurt to take longer (I think).
Counterargument: we have been following the lengths in several published papers, so either they are all mistaken / lying or this is not the problem.
2d. Wrong DAE temperatures.
No two papers use the same DAE temperatures either.
Resolution: Actually D is always 94 or 96; and E is always 72. A ranges between 50 and 65. Most papers use a value between 55 and 58 for A.
Counterargument: same as for "wrong DAE lengths": we've tried several A temperatures, all of which appear in published papers.
2e. Liquid is not heated / cooled properly (ie we are using the wrong sort of tube)
This should be easy to investigate, and is worth doing. We have a tiny amount of liquid and thus need to make sure that its temperature is changing even though it is suspended in mid-air and in plastic.
Resolution: Check (with a thermometer) the temperature of a liquid sample.
2f. Not adding correct chemicals.
The master mix may require other chemicals, such as magnesium chloride.
Resolution: Double check the documentation for the master mix.
2g. Samples are contaminated with chemicals which inhibit PCR
The most likely source of contamination is the extracted DNA template. We have been quite careful with the master mix and primers. The other possibility is that the oil we are using to prevent evaporation is damaging the reaction.
Resolution: Improve the rigour of DNA extraction. Test with various oil types.
3. Visualisation
I don't think we have problems with visualisation, because we can (almost) always see the DNA ladder.
3a. Wrong percent agarose in gel
The amount of agarose ranges between 0.8% and 2% depending on the paper.
Counterargument: 1% seems right, seems to be a good rule of thumb, and (most importantly) provides a good spread on the ladder.
Resolution: No action
3b. Wrong voltages for electrophoresis
The electrophoresis voltages vary dramatically between different papers.
Counterargument: the voltage only seems to affect the speed at which DNA moves (at least within the range we've been using). This won't give us problems. The ladder shows up okay.
Resolution: No action
3c. Lighting is wrong
The DNA may be so faint that we can't see it properly.
Counterargument: We can see the ladder, which has DNA bands consisting of 40ng of DNA. We should have much more than that if PCR is working correctly.
Resolution: No action
3d. Not loading the wells properly
We may not be loading the wells correctly. The wells are hard to see and we are not using loading dye.
Counterargument: We always load the ladder correctly. We can see faint DNA bands spreading away from the wells, which we take to be the primers. We see some glowing traces of DNA in the wells.
Resolution: No action
3e. Taking too long / not taking long enough during electrophoresis
People recommend anywhere between 20 minutes and three hours for electrophoresis (voltage dependent).
Counterargument: Like voltage, this only affects the speed of movement, and we can see the ladder fine.
Resolution: No action.
4. Primers
We may be doing something wrong w.r.t. primer design, storage, quantity, or the way we are using them.
