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Team:Paris Bettencourt/Notebook/Phage Sensor/Tuesday 16th July.html
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Revision as of 16:21, 5 August 2013
Phage Sensor
ASDFTuesday 16th July
We prepared a colony PCR to send the KAN for sequencing, to know exaclty the sequence of the KAN.
KEIOΔMPYRF has a deletion of PYRF to be replaced by KANWe pitched 4 singles colonies into 50µL of H2O
Boild 5 minutes at 95°C
1,5 µL of this can be used directly for PCR
PCR reaction
Keep all the regents at 4°C while preparing the mixture
| Reagent | Volume |
| JW 182 (10 uM) JW 183 (10 uM) Template DNA Quick-load Tag 2x Master Mix Nuclease free water | 0,5 µL 0,5 µL 1,5 µL 12,5 µL 10 µL |
| Total volume | 25 µL |
Thermocycle Protocol : NEB Quick-Load
| Temperature | Time | ||
| Start Cycle1 Cycle 2 Cycle 3 Finish Store |
95°C 95°C 50°C 68°C 68°C 10°C |
30 seconds 15 seconds 30 seconds 1 minute/kB 5 minutes Forever |
melt melt anneal extend - 35 cycles extend store |
