Project:Sex typing with amelogenin
General
The general plan is: obtain dna samples, extract dna, pcr dna with amelogenin primers, and then run that on agarose gel.
There is still work that could be done, checking the legals, ethics and safety.
We need to arrive at a definitive list of stuff (bill of materials) we need for the experiment, and source them.
There is also a need to figure out how long this will all take and schedule it. The overall duration from start to finish is going to be quite long.
It might help to try out some parts, such as the extraction, before hand.
Sampling
Current plan is to sterilise plastic sticks for consenting adult volunteers to rub inside their cheeks. Legally (I am so not a lawyer, and this is not legal advice), reading them a written spiel and getting their verbal agreement with witnesses and seeing them take the sample seems to be sufficient , although the suggestion has been made that a simple signed form might help avoid a possible problem with forgetful witnesses.
Extraction
There are a number of written protocols out there
This seems to be the typical hackers version [3] which is basically soap to lyse the cells and then meat tenderiser as a protease and separation using alcohol. Reagents are all commonly available, so seems safe enough.
do we also need to do DNA precipitation? [4] why?
PCR
[6] where it gives:
PCR Amplification of the Amelogenin Locus The primers used to amplify the X and Y amelogenin sequences were previously described by Nakahori and colleagues [6]. The primer sequences are as follows: primer AMXY-1F (5'-CTGATGGTTGGCCTCAAGCCTGTG-Y) primer AMXY-2R (5'-TAAAGAGATTCATTAACTTGACTG-3') PCR was conducted using the Perkin-Elmer GeneAmp | PCR System 9600 Thermal Cycler. Genomic DNA samples were amplified in a 100 txL reaction volume containing 0.2 mM of each dNTP, 50 mM KCI, 10 mM Tris.HC1 pH 8.3, 4.0 mM MgC12, 120 pmoles of each primer, and 2.5 U Taq polymerase. PCR was run for 30 cycles of 94~ for 1 rain (denature), 65~ for 2 min (anneal), and 72~ for 3 min (extend). Following amplification, the PCR products were analyzed by electrophoresis on 1.2% agarose gels run with 1 X TBE (89 mM Tris borate, 89 mM boric acid, 2 mM EDTA) or 6% nondenaturing polyacryl- amide gels run with 1 X TBE. The gels were stained with ethidium bromide and the fragments were visualized by fluorescence under ultraviolet light.
Andy suggests that we go with the instructions on our polymerase for the PCR, but I don't have those in front of me.
There is a question mark over whether we need a smaller pippette and we need tubes for the thermal cycler.
ISTR we have SYBR green to replace the ethidium bromide.
Gel electrophoresis
Eng has
1.2% agarose gels run with 1 X TBE (89 mM Tris borate, 89 mM boric acid, 2 mM EDTA)
If our dye is [7] then we need blue light (λmax = 497 nm)
It seems we need to manufacture new electrophoresis equipment or has someone taken it to be repaired? I looked into the possibility of making the gel tray as a single bent piece, but the guys I know said they couldn't do it on 5mm acetate.