Team:Paris Bettencourt/Notebook/Phage Sensor/Monday 19th August.html

Phage Sensor

ASDF

Monday 19^th August

Restreak XL-1 Blue Litmus28i, PCR, PCR purification Backbone (M13mp18), Ligation (RFP into M13mp18 backbone), Trafo of Ligation product (RFP in M13mp18 plasmid) into NEB turbo and Test Xgal

Restreak XL-1 Blue Litmus28i

PCR

SPCR5, SPCR6, 49,3°C

Reagent	Volume
	1x
Nuclease-free water	37,25µL
5x Phusion HF Buffer	10µL
10 mM dNTPs	1µL
Forward Primer (10 µM)	0.5µL
Reverse Primer (10 µM)	0.5µL
Template Plasmid	0.25µL
Phusion DNA Polymerase	0.5µL
Total Volume	50µL

Thermocycler Protocol: NEB Phusion
	Temp	Time
Start	98°C	30 sec	Melt
Cycle 1	98°C	5 sec	Melt	35 cycles
Cycle 2	40.5°C / 60°C	25 sec	Anneal
Cycle 3	72°C	30 sec per kb	Extend
Finish	72°C	5 min	Extend
Store	10°C	Forever	Store

PCR purification Backbone (M13mp18)
Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through. Place the QIAquick column back into the same tube
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 50 μl Buffer EB to the center of the QIAquick membrane and centrifuge the column for 1 min.

Ligation (RFP into M13mp18 backbone)
3ul Backbone
14 ul Insert
2ul 10x T4 DNA Ligase HC Buffer
1 ul T4 DNA Ligase

Incubate for at least 10min at 22°C


Trafo of Ligation product (RFP in M13mp18 plasmid) into NEB turbo
1) Thaw competent cells on ice.
2) Place 20 ul of cells in a pre-chilled Eppendorf tube.
3) Add 3 ul of plasmid (from Biobrick stock)
4) Mix gently by flicking the tube.
5) Chill on ice for 10 minutes.
4) Heat shock at 42 °C for 30 seconds
5) Return to ice for 2 minutes.
6) Add 200 ul LB medium and recover the cells by shaking at 37 °C.
7) Plate out the cells on LB (200ul)
8. Incubate at 37 °C. Transformants should appear within 12 hrs.

Test Xgal
3ml of Liquid culture with
1) Xgal from iGEM 2012 (Jake) (+IPTG)
2) Xgal from last week (in DMF) (+IPTG)