Team:Paris Bettencourt/Notebook/Phage Sensor/Wednesday 25th September.html

Detect

ASDF

Wednesday 25^th September

Gel of PCR, PCR (SPCR5,6) and Miniprep Cas9, M13mp18

Gel of PCR
1%, 100V, 1h

Picture: 13/09/25_SPCR2-10

PCR (SPCR5,6)

Reagent	Volume
	x1
Nuclease-free water	37.25 µl
5x Phusion HF Buffer	10 µl
10 mM dNTPs	1 µl
Forward Primer (10 uM)	1 µl
Reverse Primer (10 uM)	1 µl
Template Plasmid)	0.25 µl
Phusion DNA Polymerase	0.5 µl
Total Volume	50 µl

Thermocycler Protocol: Fermentas Phusion
	Temp	Time
Start	98°C	30 sec	Melt
Cycle 1	98°C	5 sec	Melt	35 cycles
Cycle 2		25 sec	Anneal
Cycle 3	72°C	30 sec per kb	Extend
Finish	72 °C	5 min	Extand
Store	10°C	Forever	Store

Miniprep Cas9, M13mp18
1) Pellet liquid culture (4000rpm, 10 min)
2) Discard supernatant
3) resuspend the cells in 250ul of resuspension olution
4) add 250ul of lysis solution, mix by inverting 4-6 times
5) add 350ul of neutralization solution
4) centrifuge for 5 min
5) transfer supernatant to spin column
6) centrifuge for 1 min
7) discard flow through
8) add 500 ul wash solution and centrifuge for 1 min , discard flow through(repeat this step)
9) centrifuge for 1 min to remove left over liquid
10) transfer the column on a 1.5ml tube
11) add 50ul of elution buffer and incubate for 2 min
12) centrifuge for 2 min
13) Nanodrop the concentration and freeze at -20°