User:Stuart McKellar/Notebook/Bird Sex Testing/2012/09/08: Difference between revisions
| Line 57: | Line 57: | ||
Master mix: (EACH TUBE) | Master mix: (EACH TUBE) | ||
1.5ul of 10x taq buffer | *1.5ul of 10x taq buffer | ||
1.5ul of 25mM MgCl2 | *1.5ul of 25mM MgCl2 | ||
100ng of each primer | *100ng of each primer | ||
200uM of each dNTP | *200uM of each dNTP | ||
0.5U taq | *0.5U taq | ||
250ng of DNA template | *250ng of DNA template | ||
Master mix(5x) | Master mix(5x) | ||
7.5ul of 10x taq buffer | *7.5ul of 10x taq buffer | ||
7.5ul of 25mM MgCl2 | *7.5ul of 25mM MgCl2 | ||
500ng of each primer | *500ng of each primer | ||
1000uM of each dNTP | *1000uM of each dNTP | ||
0.5U taq | *0.5U taq | ||
Top up water to 250ul | *Top up water to 250ul | ||
FINAL MEASUREMENTS | |||
*2.5ul dNTP mix | |||
*1.5ul Buffer | |||
*1.4ul each primer at original concentration 10uM | |||
*1.5ul MgCl2 | |||
*0.5ul taq for whole MM (so was 0.5ul/5 in ech tube) | |||
*10ul template DNA | |||
*6.7 H2O (was divided from 33.5/5) | |||
==Programming the Thermal Cycler== | ==Programming the Thermal Cycler== | ||
Revision as of 17:56, 8 September 2012
 Project name
|
<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
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.
| |
