User:Stuart McKellar/Notebook/Bird Sex Testing/2012/09/08: Difference between revisions
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I need to modify this a little bit because I am using Promega hot start taq. It needs an intial heat step to denature the antibody. This is 2minutes at 94-95C. | I need to modify this a little bit because I am using Promega hot start taq. It needs an intial heat step to denature the antibody. This is 2minutes at 94-95C. | ||
I ended up using the taq from fisher biotech so i used the following thermal cycler program: | |||
*94C 1 min | |||
*45->52 across 1 min -> held for a minute | |||
*72C for 2min | |||
*35 cycles. | |||
Revision as of 17:58, 8 September 2012
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Lab work for 8/9/2012
I am using 4 samples of the Diamond Doves, as they are being caught again in 4 weeks so it is OK if they don't work. Begin Lab workTransferred samples to 0.5mL tubes:
Carried out extraction according to protocol for tissue EDNA hispex kit. During the addition of the first solutions (1a & 1b) I used the pipette tip to disturb the blood. This pulled some of the fibres out from the paper that the blood was on. I don't think that this should hurt the reaction. Primer prep:
32.5nm (0.21mg) Tm=51.2C Added 325ul water to make up a 100uM/L stock soln (Taken from http://www.protocol-online.org/biology-forums/posts/23706.html) He posted the following: When I get my primer which comes as lyophilized, I resuspend it to 100 uM/L concentration. For example, for an amount of 28.5 nM of lyophilized primer (the amount is provided by the vendor), I add 285 ul water or TE buffer and get 100 uM/L stock solution. To prepare 10 uM/L (or 10 pm/ul) working solution, I take 10 ul stock solution and add to 90 ul water. As you can see, no calculations are needed. -pcrman-
32.6nm Tm=51.3C Added 326ul water Took 10ul of Primer and added to 90ul water to make a 10uM/L working solution. Making Master MixFollowing the suggested protocol from Promega Hot Start Taq: Component Final Vol Final Concentration Buffer 10ul 1X MgCl2(25mM) 2-8ul 1.0-4.0mM dNTPs 1ul 0.2mM each dNTP F primer X 0.1-1uM R primer Y 0.1-1uM Taq(5u/ul) 0.25ul 1.25u DNA Z <0.5ug/50ul Water to 50ul Made to 11x Master mix. Taq added to each tube individually. Changed my mind. Might start with the Taq from fisher biotech. Master mix: (EACH TUBE)
Master mix(5x)
FINAL MEASUREMENTS
Programming the Thermal CyclerMy protocol says to do the following: The conditions for PCR amplification were a denaturing step at 94 ºC for 1 min 30 s, 35 cycles of 95 ºC for 30 s, 52ºC for 30 s, and 72 ºC for 30 s, and final elongation at 72 ºC for 5 min. I need to modify this a little bit because I am using Promega hot start taq. It needs an intial heat step to denature the antibody. This is 2minutes at 94-95C. I ended up using the taq from fisher biotech so i used the following thermal cycler program:
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