Team:Paris Bettencourt/Notebook/Phage Sensor/Tuesday 16th July.html

Phage Sensor

ASDF

Tuesday 16^th 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 KAN
We 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