Team:Paris Bettencourt/Notebook/Phage Sensor/Wednesday 18th September.html

Phage Sensor

ASDF

Wednesday 16^th September

Ligations

Ligation

SPCR2 - Chl

Reagent	Volume
SPCR2	1 µl
Chl	2,95 µl
H2O	4.55 µl
Fermentas T4 Ligase Buffer	1.0 µl
Fermentas T4 Ligase Enzyme	0.5 µl
Total Volume	10 µl

SPCR2 - Amp

Reagent	Volume
SPCR2	1 µl
Amp	3.4 µl
H2O	4.1 µl
Fermentas T4 Ligase Buffer	1.0 µl
Fermentas T4 Ligase Enzyme	0.5 µl
Total Volume	10 µl

SPCR3 - Chl

Reagent	Volume
SPCR3	1 µl
Amp	1.79 µl
H2O	5.71 µl
Fermentas T4 Ligase Buffer	1.0 µl
Fermentas T4 Ligase Enzyme	0.5 µl
Total Volume	10 µl

SPCR3 - Amp7

Reagent	Volume
Amp	1 µl
SPCR 3	1.56 µl
H2O	5.94 µl
Fermentas T4 Ligase Buffer	1.0 µl
Fermentas T4 Ligase Enzyme	0.5 µl
Total Volume	10 µl

SPCR5 - Chl

Reagent	Volume
SPCR 5	0.5 µl
Chl	4.55 µl
H2O	3.45 µl
Fermentas T4 Ligase Buffer	1.0 µl
Fermentas T4 Ligase Enzyme	0.5 µl
Total Volume	10 µl

SPCR11 - Chl

Reagent	Volume
Chl	1 µl
SPCR11	1.3 µl
H2O	6.2 µl
Fermentas T4 Ligase Buffer	1.0 µl
Fermentas T4 Ligase Enzyme	0.5 µl
Total Volume	10 µl

SPCR2 - Amp

Reagent	Volume
Amp	1 µl
SPCR11	1.13 µl
H2O	6.37 µl
Fermentas T4 Ligase Buffer	1.0 µl
Fermentas T4 Ligase Enzyme	0.5 µl
Total Volume	10 µl

at 16° for 1h

Transformation
1) Thaw competent cells on ice (Gibson Kit competent cells)
2) Place 20 ul of cells in a pre-chilled Eppendorf tube.
3) Add 2.5 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.
Ampicillin: 15-30 minutes
Chloramphenicol: 60-120 minutes
7) Plate out the cells on selective LB (10ul)
8. Incubate at 37 °C. Transformants should appear within 12 hrs.

Gel of Ligation products
1%, 100V, 40 min