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Project:Sex typing with amelogenin: Difference between revisions
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Project:Sex typing with amelogenin (view source)
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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). | 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). | ||
= Complete protocol as of June 2012 = | |||
==Extraction== | |||
* Fill a PCR tube with 250 ul of 5% Chelex solution. | * Fill a PCR tube with 250 ul of 5% Chelex solution. | ||
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* Centrifuge and take supernatant. | * Centrifuge and take supernatant. | ||
==PCR== | |||
* Use the primers 5'-CTGATGGTTGGCCTCAAGCCTGTG-3' and 5'-TAAAGAGATTCATTAACTTGACTG-3' | * Use the primers 5'-CTGATGGTTGGCCTCAAGCCTGTG-3' and 5'-TAAAGAGATTCATTAACTTGACTG-3' | ||
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* Then 30 cycles of D = 96 C for one minute, A = 54 C for 30 seconds, E = 72 C for one minute. | * Then 30 cycles of D = 96 C for one minute, A = 54 C for 30 seconds, E = 72 C for one minute. | ||
==Electrophoresis== | |||
* Prepare a 1% agarose gel using 1X TBE and 2ul / 40ml of ethidium bromide. | * Prepare a 1% agarose gel using 1X TBE and 2ul / 40ml of ethidium bromide. | ||
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* Electrophorese at between 80v and 100v for 30 to 60 minutes. | * Electrophorese at between 80v and 100v for 30 to 60 minutes. | ||
==Visualisation== | |||
* Wear glasses and long sleeves. | * Wear glasses and long sleeves. | ||
* Visualise using a 300nm UV light held above the gel. View (or photograph) through an orange filter. | * Visualise using a 300nm UV light held above the gel. View (or photograph) through an orange filter. | ||
= Future Work = | |||
Given that we get this working, it should provide a basis for further experiments. As far as I understand it, it is possible to apply this sort of technique, with appropriate primers, to do detection of SNPs [http://en.wikipedia.org/wiki/Single-nucleotide_polymorphism] opening up the possibility of testing for something common like [http://en.wikipedia.org/wiki/RHD_%28gene%29 RHD] or [http://en.wikipedia.org/wiki/Melanocortin_1_receptor MC1R] alleles, and perhaps providing a resource for people who want to genetically test themselves for rarer things a la Katherine Aull (but it is much simpler and cheaper to get such tests run commercially, and there is a difficult ethical case to answer about providing genetic testing without proper expert medical support and interpretation of results). | Given that we get this working, it should provide a basis for further experiments. As far as I understand it, it is possible to apply this sort of technique, with appropriate primers, to do detection of SNPs [http://en.wikipedia.org/wiki/Single-nucleotide_polymorphism] opening up the possibility of testing for something common like [http://en.wikipedia.org/wiki/RHD_%28gene%29 RHD] or [http://en.wikipedia.org/wiki/Melanocortin_1_receptor MC1R] alleles, and perhaps providing a resource for people who want to genetically test themselves for rarer things a la Katherine Aull (but it is much simpler and cheaper to get such tests run commercially, and there is a difficult ethical case to answer about providing genetic testing without proper expert medical support and interpretation of results). | ||
