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 2x9 ml of liquid culture (4000 rpm, 10 min)
2) Discard supernatant
3) resuspend the cells in 250 µL of resuspension solution
4) add 250 µL of lysis solution, mix by inverting 4-6 times
5) add 350 µL 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 µL 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 1.5 ml tube
11) add 50 µL of elution buffer and incubate for 2 min
12) centrifuge for 2 min
13) Nanodrop the concentration and freeze at -20°

pSP001= pCOLA-DUET: 65 ng/ul



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