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

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(Programming the Thermal Cycler)
Current revision (01:17, 9 September 2012) (view source)
(Programming the Thermal Cycler)
 
Line 94: Line 94:
*35 cycles.
*35 cycles.
 +
==Preparing Buffer==
 +
 +
Making Buffer according to
 +
 +
8g NaOH
 +
45g Boric Acid
 +
ad 1L H2O
 +
(makes a 20x solution)
 +
 +
==Agar gel==
 +
III. Agarose Gel Electrophoresis
 +
 +
2.0% Agarose
 +
 +
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.

Current revision

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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.

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.

Preparing Buffer

Making Buffer according to

8g NaOH 45g Boric Acid ad 1L H2O (makes a 20x solution)

Agar gel

III. Agarose Gel Electrophoresis

2.0% Agarose

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.



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