Team:Paris Bettencourt/Notebook/Phage Sensor/Tuesday 30th July.html

Phage Sensor

ASDF

Tuesday 30^th July

Liquid culture, Miniprep and Glycerol stock

Liquid culture of:
pCOLA DUET (from liquid culture (29.07.13)) - 20 ml culture
pSSB1 (from liquid culture (29.07.13)) - 5ml culture
pSSB2 (from liquid culture (29.07.13)) - 5 ml culture
pBAC-BA-lacZ (from plate ) - 5 ml culture
pCRISPR (from plate ) - 5 ml culture
pCas9 (from plate ) - 5 ml culture
pCRIPSR::rpsL (from plate ) - 5 ml culture
F+ strain - (from glycerol stock Trojan Horse) - 10 ml culture
Keio Keio delta pyrF (from plate ) - 10 ml culture


Miniprep (pCOLA-DUET = sSP007) using the Thermo Scientific Miniprep Kit

1) Pellet 2x 9ml 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°

pSP001= pCOLA-DUET: 65ng/ul



Glycerol Stock
from overnight culture of MG1655-pCOLA-DUET (sSP007)
Centrifuge 4000rpm, 10 minutes,
take out liquid
resuspend cells in 0,5mL glycerol (60%) , 2mL LB
freeze in -80°C