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Team:Paris Bettencourt/Notebook/Phage Sensor/Thursday 5th September.html
From 2013.igem.org
Phage Sensor
ASDFThursday 5th September
Gel, Colony PCR, Topdown PCR and gels
GelSPCR12, SPCR13
1%, 100V, 20 min
Pictures: 13/09/05_SPCR12_corresponding primers
13/09/05_SPCR13_corresponding primers
Colony PCR
| Reagent | Volume |
| Forward Primer (10 uM)/td> | 0.5 ul |
| Reverse Primer (10 uM) | 0.5 ul |
| Template DNA (from above) | 1.5 ul |
| Quick-Load® Taq 2X Master Mix | 12.5 ul |
| Total Volume | 25 ul |
| Thermocycler Protocol: DreamTaq Green PCR MM | ||||
| Temp | Time | |||
| Start | 95°C | 1 min | Melt | |
| Cycle 1 | 95°C | 30 sec | Melt | 35 cycles |
| Cyle 2 | 52.6/46.8 °C | 30 sec | Anneal | |
| Cycle 3 | 72°C | 1 min per kb | Extend | |
| Finish | 72°C | 10 min | Extend | |
| Store | 10°C | Forever | Store |
Topdown PCR
SPCR 1,4,12,13
| Reagent | Volume |
| x1>/b> | |
| Nuclease-free water | 37.25 µl |
| 5x Phusion HF Buffer | 10 µl |
| 10 mM dNTPs | 1 µl |
| Forward Primer (10 uM) | 0.5 µl |
| Reverse Primer (10 uM) | 0.5 µl |
| Template Plasmid | 0.25 µl |
| Phusion DNA Polymerase | 0.5 µl |
| Total Volume | 50 µl |
increases every cycle 1°C, starting from 40°
Gels
1%, 100V, 20+ Min
Pictures:
13/09/05_colony PCR_pS005_C1-C8_BB verification primers
13/09/05_colony PCR_pS005_C9-C16_BB verification primers
13/09/05_colony PCR_pS006_C1-C8_BB verification primers
13/09/05_colony PCR_pS006_C9-C16_BB verification primers
13/09/05_top down PCR_SPCR1,4,12,13
