Team:Paris Bettencourt/Notebook/Phage Sensor/Tuesday 27th August.html
From 2013.igem.org
Phage Sensor
ASDFTuesday 27th August
Starting liquid cultures and Miniprep of pS.005 and pS.006.
Starting liquid culturessSP001, 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
Reagent | Volume |
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 Mix | 12,5 ul |
Nuclease-free water | 10,0 ul |
Total volume | 25 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 |