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Project:Sex typing with amelogenin: Difference between revisions
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(Updates now that this is mostly working) |
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We've done several more tests since March 22, most of which seem to work. 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. 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. | |||
* Scrape cheek cells using a pipette tip. | |||
* Stir the pipette tip in the Chelex so that the cells are transferred. Perhaps also spit down the pipette tip. | |||
* Incubate at 56 C for 30 minutes. Vortex for a few seconds. Incubate at 94 C for 10 minutes. | |||
* Centrifuge and take supernatant. | |||
'''PCR''': | |||
* Use the primers 5'-CTGATGGTTGGCCTCAAGCCTGTG-3' and 5'-TAAAGAGATTCATTAACTTGACTG-3' | |||
* Use 12.5 ul of Taq master mix, 2 ul of each primer, 3 ul of template, and 5.5 ul of distilled water. | |||
* Initial denat. of 96 C for 5 minutes. | |||
* 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. | |||
* Electrophorese at between 80v and 100v for 30 to 60 minutes. | |||
'''Visualisation''': | |||
* Wear glasses and long sleeves. | |||
* Visualise using a 300nm UV light held above the gel. View (or photograph) through an orange filter. | |||
== Future Work == | == Future Work == | ||
