User:Stuart McKellar/Notebook/Bird Sex Testing/2012/12/01

higher annealing temp

Aim: test the p2/p8 and 2550/2178 primers at 65°C.

Method:

Sample 1:

• A6: p2/p8 primers
• B6: 2550/2178 primers

Sample 2:

• C6: p2/p8 primers
• D6: 2550/2178 primers

Negative control - no DNA (1ul h2O, 2550/2178 primers.)

Master mix (5x):

• 12.5μL buffer 10x
• 12.5μL dNTPS
• 7.5μL MgCl₂
• 62.5 H₂O
• 1μL Taq

• A6: p2/p8 primers (2.5μL each, 1μL DNA) + 18.5 Master Mix
• B6: 2550/2178 primers (2.5μL each, 1μL DNA) + 18.5 Master Mix
• C6: p2/p8 primers (2.5μL each, 1μL DNA) + 18.5 Master Mix
• D6: 2550/2178 primers (2.5μL each, 1μL DNA) + 18.5 Master Mix

total volume in each is 25μL.

PCR: 94°C for 2m30s, 35 cycles of: 95°C 30s, 65°C for 30s, 72°C for 30s; final elongation 72°C for 5 mins( actually went 10 mins by accident).

results to be determined via agar gel electrophoresis on 1% gel stained using gelgreen.