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Team:Paris Bettencourt/Notebook/Phage Sensor/Wednesday 7th August.html
From 2013.igem.org
Phage Sensor
ASDFWednesday 7th August
Glycerol Stock of sSP008-sSP0011, M13 Test, Single Colonies, Liquid cultures, PCR and Transformation
Glycerol Stock of sSP008-sSP0011
Take 1.5ml of ON culture + 500ul Glycerol (60%)
Freeze at -80°C
M13 Test
Plate from ON culture:
1) 1:1000 - sSP012: sSP009 (plate 200ul)
2) 200 ul sSP012
3) 200ul sSp009
Single Colonies
streak out ON culture of sSP012 to get single colonies
Liquid cultures of pir+ E.coli and pKD13 from Jake
PCR of SPCR4, SPCR7, SPCR8, SPCR9, SPCR10 from circuar backbone, directly taken from Biobrick plate
Protocol
| 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 |
Transformation Biobricks BBa_K649304 (with pSB1C3 backbone) and BBa_J04450 (with pSB1A3 backbone) into NEB Turbo (heat shock trafo)
1) Thaw competent cells on ice.
2) Place 20 ul of cells in a pre-chilled Eppendorf tube.
3) Add 2.5 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.
Ampicillin: 15-30 minutes
Chloramphenicol: 60-120 minutes
7) Plate out the cells on selective LB (10ul)
8) Incubate at 37 °C. Transformants should appear within 12 hrs.
Result: BBa_J04450: many colonies, BBa_K649304 no colonies, plate rest
