Project:Sex typing with amelogenin: Difference between revisions

The general plan, with thanks to Maria, is: obtain dna samples, extract dna, pcr dna with amelogenin primers, and then run that on agarose gel.

It might help to try out some parts, such as the extraction, before hand, although I don't see how we know whether that works until we run it through the rest. We could practise making gels, running a ladder on them, to figure out mixtures and timings and practise with detecting the dye.

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.

cotton swabs are another possible option.

There are a number of written protocols out there

The sampling and extraction are crucial steps if the following steps are to work. dna contamination can be amplified in the pcr stage. there is a suggestion that there is an amount of black art in getting this to work well. It might repay the effort to try several different extractions and run them side by side for comparison.

We have SYBR green to replace the ethidium bromide.

Eng has  

[http://www.shef.ac.uk/content/1/c6/06/14/01/CoSHH%20059_PCR-TGGE%20and%20geldoc.pdf Example COSHH for PCR and gel electrophoresis with TAE]

[http://www.nbsbio.co.uk/product.asp?pID=2490&c=283957 SafeView] and [http://www.nbsbio.co.uk/product.asp?pID=6241&cID=71 SafeWhite] are from [http://www.nbsbio.co.uk/ NBS Bio]. There are manuals and safety sheets there.

interesting doc about ethidium bromide disposal in th uk here: [http://www.docs.csg.ed.ac.uk/estatesbuildings/waste/Ethidium_Bromide_briefing.pdf]

We expect bands at X = 977 bp and Y = 788 bp

It seems that the lines at X=977bp are in different positions in the male and female lanes in the cybertory example. discuss.

Individuals with deletions such that this test doesn't work on them are extremely rare. So we hope to get correct results for all samples. We discussed blinding the samples to avoid possible bias in interpretation reading the gel, but if we can photograph the gel that could serve that purpose.  

It might be nice to do video recording and a write up.

On this date, we had not yet received our PCR tubes, so we were unable to do PCR. Instead, we decided to test our ability to do electrophoresis, using a DNA ladder and SafeWhite stain. We followed the following protocol:

We resolved to make the next test a simple visualisation test: combine ladder with Safewhite and attempt to visualise under UV.

Nicholas combined 2.5ul SafeWhite and 2.5ul DNA Marker-D. He was unable to make this mixture glow under UV.

Nicholas performed the same protocol as in the "First gel run, Dec 7, 2011", with the following changes:

  * No staining was observed.

We successfully visualised a ladder using the following protocol:

* Visualised by placing a fluorescent tube with peak emission at 300 nm directly underneath the gel.

our Technical datasheet gives numbers for water to add to rehydrate our primers to 100uM.

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File:Gel_IMG_1081_rotated.JPG|Nice picture

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We've done several more tests since March 22, most of which seem to work. Summaries [http://wiki.london.hackspace.org.uk/view/Sex_typing_with_amelogenin/Results-latest here] The major problem seems to be quality of the extracted DNA: PCR product is frequently quite faint. As of April 26, 2012, DNA extraction technique will be the main focus for the amelogenin test (and for genetic testing of human DNA in general).

'''Extraction''':