User:Stuart McKellar/Notebook/Bird Sex Testing/2012/12/10
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(Autocreate 2012/12/10 Entry for User:Stuart_McKellar/Notebook/Bird_Sex_Testing) |
Current revision (22:53, 10 December 2012) (view source) (→Entry title) |
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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. | ||
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| + | 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. | ||
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| + | repeated experiment at 58°C. | ||
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| + | ran PCR for 35 cycles, annealing temperature is 60°C | ||
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| + | 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. | ||
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| + | 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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| + | *****DO SOME READING OF THE PAPERS AFTER PCR HAS STARTED***** | ||
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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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