User:Stuart McKellar/Notebook/Bird Sex Testing/2012/09/08

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(Begin Lab work)
(Making Master Mix)
Line 41: Line 41:
==Making Master Mix==
==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)
 +
1.5ul of 10x taq buffer
 +
1.5ul of 25mM MgCl2
 +
100ng of each primer
 +
200uM of each dNTP
 +
0.5U taq
 +
250ng of DNA template
 +
 +
Master mix(5x)
 +
7.5ul of 10x taq buffer
 +
7.5ul of 25mM MgCl2
 +
500ng of each primer
 +
1000uM of each dNTP
 +
0.5U taq
 +
Top up water to 250ul
 +
 +
==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.

Revision as of 07:01, 8 September 2012

Project name Main project page
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Lab work for 8/9/2012

  • I am going to attempt the extraction of DNA from some samples using the EDNA extraction kit and protocol. If this is successful I will go on to run a PCR and a Gel.

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:

  • 6AMC
  • L3#29
  • 8AMC
  • L3#21

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:

  • 2550F - 5´-GTTACTGATTCGTCTACGAGA-3´

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-


  • 2718R - 5´-ATTGAAATGATCCAGTGCTTG-3´

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) 1.5ul of 10x taq buffer 1.5ul of 25mM MgCl2 100ng of each primer 200uM of each dNTP 0.5U taq 250ng of DNA template

Master mix(5x) 7.5ul of 10x taq buffer 7.5ul of 25mM MgCl2 500ng of each primer 1000uM of each dNTP 0.5U taq Top up water to 250ul

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.




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