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V08_13
2nd round: Mutagenesis PCR, 1000 µL
- Experiment:
The PCR was set up in a volume of 1000 µL following the protocol from week 10.
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V08_14
2nd round: PCR purification
- Experiment:
The PCR purification was performed using the peqGOLD Cycle-pure Kit (PeqLab), modified using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab)! Elution in 100 µL EB.
- Observations & Results:
We now seemed to have found the mistake that caused problems with the loss of DNA material in the second mutagenesis round! Miscommunication led to using the wrong kit for purification steps. We decided to use the peqGOLD Cycle-pure Kit (PeqLab) instead of the subsequent ethanol precipitation as well, as this seemed to rise fewer problems.
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V08_15
V08_15_1 2nd round: DpnI/BsaI digest and purification
- Experiment:
The digest was performed according to protocol from week 10. The subsequent purification was carried out using the peqGOLD Cycle-pure Kit (PeqLab)!!!
- Observations & Results:
The corresponding agarose gel showed band of the expected sizes! At last, there did not seem to be any loss of DNA material.
V08_15_2 2nd round: Ligation, 2000 µL
- Experiment:
The ligation was performed according to the establoshed protocol from week 10. Incubation over night at 16 °C.
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V08_16
V08_16_1 2nd round: Purification of ligation from V08_15
- Experiment:
The purification of the ligation was now done using the peqGOLD Cycle-pure Kit (PeqLab) with columns from peqGOLD Plasmid Miniprep Kit I (PeqLab) instead of an ethanol precipitation. This way we could be sure to gain enough DNA material to go on with our library generation.
- Observations & Results:
We now seemed to have found the mistake that caused problems with the loss of DNA material in the second mutagenesis round! Miscommunication led to using the wrong kit for purification steps. We decided to use the peqGOLD Cycle-pure Kit (PeqLab) instead of the subsequent ethanol precipitation as well, as this seemed to rise fewer problems.
V08_16_2 2nd round: Ligation
- Experiment:
The PCR purification was performed using the peqGOLD Cycle-pure Kit (PeqLab), modified using columns from the peqGOLD Plasmid Miniprep Kit I (PeqLab)! Elution in 100 µL EB.
- Observations & Results:
We now seemed to have found the mistake that caused problems with the loss of DNA material in the second mutagenesis round! Miscommunication led to using the wrong kit for purification steps. We decided to use the peqGOLD Cycle-pure Kit (PeqLab) instead of the subsequent ethanol precipitation as well, as this seemed to rise fewer problems.
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V08_17
V08_17_1 2nd round: DpnI/BsaI digest and purification
- Experiment:
The PCR purification was performed using the peqGOLD Cycle-pure Kit (PeqLab), modified using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab)! Elution in 100 µL EB.
- Observations & Results:
We now seemed to have found the mistake that caused problems with the loss of DNA material in the second mutagenesis round! Miscommunication led to using the wrong kit for purification steps. We decided to use the peqGOLD Cycle-pure Kit (PeqLab) instead of the subsequent ethanol precipitation as well, as this seemed to rise fewer problems.
V08_17_2 2nd round: Ligation
- Experiment:
The PCR purification was performed using the peqGOLD Cycle-pure Kit (PeqLab), modified using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab)! Elution in 100 µL EB.
- Observations & Results:
We now seemed to have found the mistake that caused problems with the loss of DNA material in the second mutagenesis round! Miscommunication led to using the wrong kit for purification steps. We decided to use the peqGOLD Cycle-pure Kit (PeqLab) instead of the subsequent ethanol precipitation as well, as this seemed to rise fewer problems.
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