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

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