Team:Paris Bettencourt/Notebook/Phage Sensor/Wednesday 31st July.html

Phage Sensor

ASDF

Wednesday 31^st July

Conjugation, Miniprep addgene plasmids,Glycerol Stock of addgene plasmids

Conjugation

sSP001, SP002, keio Delta PyrF
F+ comes from XL1 Blue (glycerol stock Trojan Horse) => F+ is tetR

Protocol :
1) From O/N cultures Dilute strains 1/100 in LB
2) Wait for OD to reach O,2
3) Prepare 4 tubes (in BD tubes) :
- Tube 1 = 0,5mL LB with Strain 1 (sSP001) ,5mL LB = control
- Tube 2 = 0,5mL LB with Strain 2 (sP001) + 0,5mL LB = control
- Tube 3 = 0,5mL LB with Strain 3 (keio Delta pyrF) + 0,5mL LB = control
- Tube 4 = 0,5mL LB with Strain 4 (XL1) + 0,5mL LB = control
- Tube 5 = 0,5mL LB with Strain 1 (sSP001) + 0,5mL LB with Strain 4 (XL1)
- Tube 6 = 0,5mL LB with Strain 2 (sSP002) + 0,5mL LB with Strain 4 (XL1)
- Tube 7 = 0,5mL LB with Strain 3 (keio Delta pyrF) + 0,5mL LB with Strain 4 (XL1)
4) Incubate 2 hours at 37°C (actually not in the shaker, but we accidently kept them in the shaker...)
5) Plate 10ul for controls, 100uL, 10ul for mixed tubes on LB antiobiotics (Tube 5: Chl + Tet, Tube 6: Chl + Tet, Tube 7: Kan + Tet)
6) Incubate overnight at 37°C

Miniprep addgene plasmids
1) Pellet 4ml of liquid culture (4000rpm, 10 min)
2) Discard supernatant
3) resuspend the cells in 250ul of resuspension olution
4) add 250ul of lysis solution, mix by inverting 4-6 times
5) add 350ul of neutralization solution
4) centrifuge for 5 min
5) transfer supernatant to spin column
6) centrifuge for 1 min
7) discard flow through
8) add 500 ul wash solution and centrifuge for 1 min , discard flow through(repeat this step)
9) centrifuge for 1 min to remove left over liquid
10) transfer the column on a 1.5ml tube
11) add 50ul of elution buffer and incubate for 2 min
12) centrifuge for 2 min
13) Nanodrop the concentration and freeze at -20°

See Database for plasmid reference
pCas9:
pCRISPR:
pBac-LacZ:
pCRISPR::rpsL:

Glycerol Stock of addgene plasmids
(see Database for strain reference)
from overnight culture
Centrifuge 4000rpm, 10 minutes,
take out liquid
resuspend cells in 0,5mL glycerol (60%) , 2mL LB
freeze in -80°C