Team:Paris Bettencourt/Notebook/Phage Sensor/Monday 19th August.html
From 2013.igem.org
Phage Sensor
ASDFMonday 19th August
Restreak XL-1 Blue Litmus28i, PCR, PCR purification Backbone (M13mp18), Ligation (RFP into M13mp18 backbone), Trafo of Ligation product (RFP in M13mp18 plasmid) into NEB turbo and Test Xgal
Restreak XL-1 Blue Litmus28iPCR
SPCR5, SPCR6, 49,3°C
Reagent | Volume |
1x | |
Nuclease-free water | 37,25µL |
5x Phusion HF Buffer | 10µL |
10 mM dNTPs | 1µL |
Forward Primer (10 µM) | 0.5µL |
Reverse Primer (10 µM) | 0.5µL |
Template Plasmid | 0.25µL |
Phusion DNA Polymerase | 0.5µL |
Total Volume | 50µL |
Thermocycler Protocol: NEB Phusion | ||||
Temp | Time | |||
Start | 98°C | 30 sec | Melt | |
Cycle 1 | 98°C | 5 sec | Melt | 35 cycles |
Cycle 2 | 40.5°C / 60°C | 25 sec | Anneal | |
Cycle 3 | 72°C | 30 sec per kb | Extend | |
Finish | 72°C | 5 min | Extend | |
Store | 10°C | Forever | Store |
PCR purification Backbone (M13mp18)
Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through. Place the QIAquick column back into the same tube
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 50 μl Buffer EB to the center of the QIAquick membrane and centrifuge the column for 1 min.
Ligation (RFP into M13mp18 backbone)
3ul Backbone
14 ul Insert
2ul 10x T4 DNA Ligase HC Buffer
1 ul T4 DNA Ligase
Incubate for at least 10min at 22°C
Trafo of Ligation product (RFP in M13mp18 plasmid) into NEB turbo
1) Thaw competent cells on ice.
2) Place 20 ul of cells in a pre-chilled Eppendorf tube.
3) Add 3 ul of plasmid (from Biobrick stock)
4) Mix gently by flicking the tube.
5) Chill on ice for 10 minutes.
4) Heat shock at 42 °C for 30 seconds
5) Return to ice for 2 minutes.
6) Add 200 ul LB medium and recover the cells by shaking at 37 °C.
7) Plate out the cells on LB (200ul)
8. Incubate at 37 °C. Transformants should appear within 12 hrs.
Test Xgal
3ml of Liquid culture with
1) Xgal from iGEM 2012 (Jake) (+IPTG)
2) Xgal from last week (in DMF) (+IPTG)