User:Stuart McKellar/Notebook/Bird Sex Testing/2012/11/23: Difference between revisions
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== | ==PCR optimisation == | ||
* | Yesterday failed to yield a single successful result. The two steps that were universal across all samples were number of cycles, an extended 72C period of 20 mins after 25 cycles, and 0.5U taq. | ||
Aim: Today I will try with 3 different levels of taq and dna and run everything else the same as for the initial successful run. The tubes contents will be as follows: | |||
*1 x Master Mix(uL): | |||
*2.5 Buffer (10x) | |||
*2.5 Forward (P2) | |||
*2.5 Reverse (P8) | |||
*2.5 dNTPs | |||
*1.5 MgCl2 | |||
*10 H20 | |||
DNA From R5#9 (DNA 186.53 ng/ul) | |||
Then as follows for each tube: | |||
*AA: 0.5ul DNA, 0.5U Taq; H20= 2.5 | |||
*AB: 0.5ul DNA, 1.0U Taq; H20= 2.0 | |||
*AC: 0.5ul DNA, 1.5U Taq; H20= 1.5 | |||
*AD: 1.0ul DNA, 0.5U Taq; H20= 2.0 | |||
*AE: 1.0ul DNA, 1.0U Taq; H20= 1.5 | |||
*AF: 1.0ul DNA, 1.5U Taq; H20= 1.0 | |||
*AG: 2.0ul DNA, 0.5U Taq; H20= 1.0 | |||
*AH: 2.0ul DNA, 1.0U Taq; H20= 0.5 | |||
*AI: 2.0ul DNA, 1.5U Taq; H20= 0.0 | |||
*A-ve: 0.0ul DNA 1.0U Taq; H20= 2.5 | |||
All of the tubes had 24.5 ul total. They had 21ul of MM, instead of 21.5. | |||
Visualised on a 1% gel. 500V, 75mA for about 30 minutes. Bars everywhere! Sample looks female. | |||
Revision as of 18:35, 26 November 2012
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PCR optimisationYesterday failed to yield a single successful result. The two steps that were universal across all samples were number of cycles, an extended 72C period of 20 mins after 25 cycles, and 0.5U taq. Aim: Today I will try with 3 different levels of taq and dna and run everything else the same as for the initial successful run. The tubes contents will be as follows:
DNA From R5#9 (DNA 186.53 ng/ul) Then as follows for each tube:
All of the tubes had 24.5 ul total. They had 21ul of MM, instead of 21.5.
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