Team:Paris Bettencourt/Notebook/Phage Sensor/Monday 16th September.html

Phage Sensor

ASDF

Monday 16^th September

Result Cloning, Protocol: E. coli Colony PCR, Extraction of DNA, PCR Reaction and Gels

Result Cloning:
Colonies?
pS002 (Chl): no
pS002 (Amp): yes
pS003 (Amp):yes
pS004 (Chl): no
pS004 (Amp): yes
pS005 (Chl): yes
pS006-7 (Chl): yes
pS006 (Chl): no
pS007 (Chl): yes
pS008 (Amp): yes
pS009 (Chl): yes
pS012 (Amp): yes
pS013 (Chl): no
pS013 (Amp): yes

Protocol: E. coli Colony PCR
Using the Dream Taq Master Mix

Extraction of DNA

1) Pick a single colony into 50 ul of H20.

2) Boil for 5 minutes.

PCR Reaction (pS002 (Chl-Lig), pS005 (Chl-G), pS006-7 (Chl-Lig), pS007 (Chl-G), pS008 (Amp-G), pS009 (Chl-G))

Keep all the reagents at 4 °C while preparing the mixture.
Pre-heat the thermocycler to 95 °C and transfer your reaction directly from 4 °C.

Reagent	Volume
Forward Primer (10 uM)	0.5 ul
Reverse Primer (10 uM)	0.5 ul
Template DNA (from above)	1.5 ul
Quick-Load® Taq 2X Master Mix	12.5 ul
Nuclease-free water	10 ul
Total Volume	25 ul

Thermocycler Protocol: NEB Quick-Load
	Temp	Time
Start	95°C	30 sec	Melt
Cycle 1	95°C	15 sec	Melt	35 cycles
Cycle 2	45.8 °C	30 sec	Anneal
Cycle 3	72°C	1 min per kb	Extend
Finish	72 °C	15 min	Extand
Store	10°C	Forever	Store

Gels
100V, 25-30 min, 1%

Pictures:
13/09/16_colony PCR_ps007.BMP
13/09/16_colony PCR_pS006-7.BMP
13/09/16_colony PCR_ps005.BMP

Result: didn’t work