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Team:Paris Bettencourt/Notebook/Phage Sensor/Friday 16th August.html
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sSP017<br> | sSP017<br> | ||
<br> | <br> | ||
| - | 1) Pellet 2x | + | 1) Pellet 2x 2ml of liquid culture (4000rpm, 10 min)<br> |
2) Discard supernatant<br> | 2) Discard supernatant<br> | ||
3) resuspend the cells in 250ul of resuspension olution<br> | 3) resuspend the cells in 250ul of resuspension olution<br> | ||
Latest revision as of 10:09, 29 August 2013
Phage Sensor
ASDFFriday 16th August
Liquid M13 Test, Glycerol Stock, Miniprep
Liquid M13 TestOD (sSP020=XL10 KanR with F+)=0,557
OD (sSP016=XL10 KanR)=0,515
OD (sSP009= delta pyrF with F+)=0,561
OD(sSP012=FR-433=M13)=0,695
Prepare Falcons containing 500ul of each strain, adding 500ul of 10^-6 dilution of FR-433
add 1ul of IPTG and 1ul of Xgal
Incubate at 37° on shaker
Glycerol Stock sSP019 (pir+ E.coli transformed with pKD13)
sSP020 (XL10 KanR with F+)
Miniprep
sSP019
sSP017
1) Pellet 2x 2ml 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°
pKD13: 311ng/ul
Litmus 28i: 93ng/ul
