User:Stuart McKellar/Notebook/Bird Sex Testing/2012/09/08
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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 work
Transferred 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.
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 Mix
Following 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)
Programming the Thermal Cycler
My 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:
Making Buffer according to
8g NaOH 45g Boric Acid ad 1L H2O (makes a 20x solution)
III. Agarose Gel Electrophoresis
Makes 200 ml. Use fresh or store jelled at room temperature (several weeks).
Add 4.0 g agarose (electrophoresis grade) to 200 ml 1X TBE electrophoresis buffer in a 600 ml beaker or Erlenmeyer flask. Stir to suspend agarose. Cover beaker with aluminum foil, and heat in boiling-water bath (double boiler) or on hot plate until all agarose is dissolved (approximately 10 minutes). or Heat uncovered in a microwave oven at high setting until all agarose is dissolved (3-5 minutes per beaker). Swirl solution and check bottom of beaker to insure that all agarose has dissolved. (Just prior to complete dissolution, particles of agarose appear as translucent grains.) Reheat for several minutes if necessary. Cover with aluminum foil, and hold in a hot-water bath (at about 60°C) until ready for use. Remove any "skin" of solidified agarose from surface prior to pouring.