Team:Paris Bettencourt/Notebook/Phage Sensor/Tuesday 13th August.html

Phage Sensor

ASDF

Tuesday 13^th August

PCR circular and linear 40.5°C/ 60°C, Conjugation of XL-10 (with sSP011), Patches, Digestion M13 backbone, RFP insert and Gel of PCR

PCR circular and linear, 40.5°C, 60° of pSB1A3, pSB1C3

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

Conjugation of XL-10 (with sSP011)
1) From O/N cultures Dilute strains 1/100 in LB
2) Wait for OD to reach O,2
3) Prepare tube (in BD tubes) :
- Tube = 0,5mL LB with Strain (sSP011) + 0,5mL LB with Strain (XL-10 Kan)
4) Incubate 2 hours at 37°C (actually not in the shaker, but we accidently kept them in the shaker...)
5) Plate 20ul for mixed tube on LB antiobiotics (Tet, Kan)
6) Incubate overnight at 37°C


Patches
check and make new ones


Digestion M13 backbone, RFP insert
for Backbone (M13mp18 plasmid): 3ug
7,58 ul plasmid (c=395ng/ul)
3 ul EcoRI
3 ul PstI
3ul 10x Fast Digest
13,42 ul H20
incubate for 12 min on 37°
heat inactivation: 80° 5 min
for insert (BBa_J04450): 5ug
31,4 ul plasmid
5 ul EcoRi
5 ul PstI
3,6 ul H20
incubate for 20 min at 37°
heat inactivation: 80° 5min

Gel of PCR (Amp 40.5 circ, lin, Chl 60° lin, circ)
100V, 20 min 1% gel