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


Phage Sensor

Tuesday 13th 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

Nuclease-free water37,25µL
5x Phusion HF Buffer10µL
10 mM dNTPs1µL
Forward Primer (10 µM)0.5µL
Reverse Primer (10 µM)0.5µL
Template Plasmid0.25µL
Phusion DNA Polymerase0.5µL
Total Volume50µL

Thermocycler Protocol: NEB Phusion
Start98°C30 secMelt
Cycle 198°C5 secMelt35 cycles
Cycle 240.5°C / 60°C25 secAnneal
Cycle 372°C30 sec per kbExtend
Finish72°C5 minExtend

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

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