User:Stuart McKellar/Notebook/Bird Sex Testing/2012/12/10: Difference between revisions
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== | ==PCR with positive controls== | ||
* | |||
Aim: See if we can identify the positive controls using both primers. | |||
Materials and methods: We will be running 5 x PCR reactions: male and female with p2/p8, male and female with 2550/2178 primers, and a negative control. We will run it at 60°C. | |||
reaction mix: | |||
*2.5ul F primer | |||
*2.5ul R primer | |||
*2.0ul dNTPs | |||
*0.5u Taq | |||
*1ul DNA | |||
*2.5ul 10x buffer | |||
*1.5ul MgCl<sub>2</sub> | |||
*13ul H<sub>2</sub>O | |||
Master Mix: | |||
12.5 F | |||
12.5 R | |||
0.5 Taq | |||
10 dNTPs | |||
12.5 Buffer | |||
7.5 MgCl<sub>2</sub> | |||
65ul H<sub>2</sub>O | |||
Negative control used p2/p8 primers as they are the most likely ones to work at these temps. Primers were not added to MM, but instead to each individual tube. | |||
repeated experiment at 58°C. | |||
ran PCR for 35 cycles, annealing temperature is 60°C | |||
looks like faint bands are appearing. I may need to alter the times a little bit, maybe extend the amount of time for elongation. Two bands were distinguishable on the 58 and 60C PCRs when viewed on a 1% gel. | |||
The second one which was 58C annealing temp ran on a 1% gel. I want to try 2550 primer set at 55C today. I might run the 2550 samples on the tawny's and the eclectus. | |||
*****DO SOME READING OF THE PAPERS AFTER PCR HAS STARTED***** | |||
Revision as of 19:53, 10 December 2012
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PCR with positive controlsAim: See if we can identify the positive controls using both primers. Materials and methods: We will be running 5 x PCR reactions: male and female with p2/p8, male and female with 2550/2178 primers, and a negative control. We will run it at 60°C. reaction mix:
Master Mix: 12.5 F 12.5 R 0.5 Taq 10 dNTPs 12.5 Buffer 7.5 MgCl2 65ul H2O Negative control used p2/p8 primers as they are the most likely ones to work at these temps. Primers were not added to MM, but instead to each individual tube. repeated experiment at 58°C. ran PCR for 35 cycles, annealing temperature is 60°C looks like faint bands are appearing. I may need to alter the times a little bit, maybe extend the amount of time for elongation. Two bands were distinguishable on the 58 and 60C PCRs when viewed on a 1% gel. The second one which was 58C annealing temp ran on a 1% gel. I want to try 2550 primer set at 55C today. I might run the 2550 samples on the tawny's and the eclectus.
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