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Team:Wageningen UR/Journal/week7
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*Prepare sample for PCR | *Prepare sample for PCR | ||
| - | *Run PCR (98°C for 10 seconds, 55 °C for 30 seconds, 72 °C for 30 seconds, 35 cycles, | + | *Run PCR (98°C for 10 seconds, 55 °C for 30 seconds, 72 °C for 30 seconds, 35 cycles, Phusion enzyme) |
! due to unknown reasons the standard program was altered, which resulted into a temperature gradient of the annealing temperature from 52- 58 °C. All the products were in the 58 °C region of the temperature gradient | ! due to unknown reasons the standard program was altered, which resulted into a temperature gradient of the annealing temperature from 52- 58 °C. All the products were in the 58 °C region of the temperature gradient | ||
*Prepare PCR-samples for agarose gel | *Prepare PCR-samples for agarose gel | ||
| Line 39: | Line 39: | ||
Tuesday: | Tuesday: | ||
| - | *Gradient PCR was performed for the CCMV unmodified gene amplification. Annealing temperature will have a gradient of 50-55 degrees, as temperatures above did not observe desired results. The other settings were the same as de PCR of | + | *Gradient PCR was performed for the CCMV unmodified gene amplification. Annealing temperature will have a gradient of 50-55 degrees, as temperatures above did not observe desired results. The other settings were the same as de PCR of Monday. |
*All PCR reactions gave a positive result, with the best result at 53 degrees Celcius. | *All PCR reactions gave a positive result, with the best result at 53 degrees Celcius. | ||
Latest revision as of 22:55, 26 September 2012
week 7: 11 June - 19 June
Office work
Wiki page
Monday:
- Update protocol and journal
Other
Tuesday:
- Preparations made for moving to a larger lab
[Meeting]
written by: Mark
Lab work
Bricking of CCMV and d26-CCMV second try
Monday:
- Prepare sample for PCR
- Run PCR (98°C for 10 seconds, 55 °C for 30 seconds, 72 °C for 30 seconds, 35 cycles, Phusion enzyme)
! due to unknown reasons the standard program was altered, which resulted into a temperature gradient of the annealing temperature from 52- 58 °C. All the products were in the 58 °C region of the temperature gradient
- Prepare PCR-samples for agarose gel
- Run agarose gel (1% w/v agarose)
- Check gel
All products amplified well, except for the CCMV unmodified product. Investigation gives the suspicion that the annealing temperature was to high. Gradient PCR will be performed to find out the right annealing temperature for CCMV.
Tuesday:
- Gradient PCR was performed for the CCMV unmodified gene amplification. Annealing temperature will have a gradient of 50-55 degrees, as temperatures above did not observe desired results. The other settings were the same as de PCR of Monday.
- All PCR reactions gave a positive result, with the best result at 53 degrees Celcius.
Testing CCMV protocol
11 June (Mark)
- Prepare samples for ultracentrifuge according to protocol
- Run ultracentrifuge
