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

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(Making Master Mix)
(Making Master Mix)
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 20:56, 8 September 2012

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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

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

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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