Team:Paris Bettencourt/Notebook/Phage Sensor/Tuesday 27th August.html

From 2013.igem.org

Phage Sensor

Tuesday 27th August

Starting liquid cultures and Miniprep of pS.005 and pS.006.

Starting liquid cultures
sSP001, sSP002, sSP010, sSP011, sSP012, sSP020

Miniprep of pS.005 and pS.006
1) Pellet 2x 9ml of liquid culture (4000rpm, 10 min)
2) Discard supernatant
3) resuspend the cells in 250ul of resuspension solution
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°

Colony PCR

ReagentVolume
Forward Primer (10 uM)0,5 ul
Reverse Primer (10 uM)0,5 ul
Template DNA (Miniprep)0,5 ul
Quick-Load® Taq 2X Master Mix12,5 ul
Nuclease-free water10,0 ul
Total volume25 ul

Thermocycler Protocol: Dream Taq Green
Temp Time
Start 95°C 30 sec1 Melt
Cycle 1 95°C 15 sec Melt 35 cycles
Cycle 2 46,8°C 30 sec Anneal
Cycle 3 68°C 1 min per kb Extend cellule 2
Finish 68°C 5 min Extend
Store 10°C Forever Store