Team:Paris Bettencourt/Notebook/Phage Sensor/Monday 30th September.html
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Detect
ASDFMonday 30th September
Extraction of Genomic DNA, PCR reaction, Gels, Miniprep, PCR: SPCR5, SPCR6 and Liquid culture of pS004’ and pS013’
Protocol: E. coli Colony PCR (pS006, pS013’,pS013*, pS004’, pS002’)Extraction of Genomic DNA
1) Pick a single colony into 50 ul of H20.
Fresh colonies (grown that day) work best, but they can also come from 4 C.
Pipet 2ul onto plates to have C1-C8
2) Boil for 5 minutes.
1.5 ul of this can be used directly for PCR.
Best if used directly, but can also be stored at 4 C for a few days.
PCR Reaction
Keep all the reagents at 4 °C while preparing the mixture.
Pre-heat the thermocycler to 95 °C and transfer your reaction directly from 4 °C.
Reagent | Volume | 9x Volume |
Forward Primer (10 uM) | 0.5 µl | 4.5 µl |
Reverse Primer (10 uM) | 0.5 µl | 4.5 µl |
Template DNA (from above) | 1.5 µl | 13.5 µl |
Quick-Load® Taq 2X Master Mix | 12.5 µl | 112.5 µl9x Volume |
Nuclease-free water | 10 µl | 90 µl |
Total Volume | 25 µl | 125 µl |
Thermocycler Protocol: Green Dream Taq | ||||
Temp | Time | |||
Start | 95°C | 30 sec | Melt | |
Cycle 1 | 95°C | 15 sec | Melt | 35 cycles |
Cycle 2 | 46.8°C | 30 sec | Anneal | |
Cycle 3 | 72°C | 1 min per kb | Extend | |
Finish | 72 °C | 10 min | Extand | |
Store | 10°C | Forever | Store |
Gels
1%, 100V, 45 min
Pictures + Results
13/09/30_colony pcr_pS004_pS013 - 1-8: pS004 (gRNA - Amp), 9-16: pS013 (crRNA - Amp) - seems to have worked beside pS013 C7
![](https://static.igem.org/mediawiki/2013/c/c5/13-09-30_colony_pcr_pS004_pS013.png)
13/09/30_colony PCR_pS004*_pS002* - 1-8: pS004* (gRNA - Amp), 9-16: pS002 (reporter - Amp) - pS004* seems to have worked (C1, C2, C3, C6)
![](https://static.igem.org/mediawiki/2013/2/20/3-09-30_colony_PCR_pS004%2A_pS002%2A.png)
13/09/30_colony PCR_pS006'_pS006* - 1-8: pS006' (Cas9 - Chl), 9-16: pS006* (Cas9 - Amp) - didn’t work
![](https://static.igem.org/mediawiki/2013/4/44/13-09-30_colony_PCR_pS006%27_pS006%2A.png)
13/09/30_Colony PCR_pS013'_pS013* - 1-8: pS013' (crRNA - Chl), 9-16: pS013* (crRNA - Amp) - Seems to have worked beside 13’ C7, C8
![](https://static.igem.org/mediawiki/2013/c/ca/13-09-30_Colony_PCR_pS013%27_pS013%2A.png)
13/09/30_colony PCR_pS004'_pS002' - 1-8: pS004' (gRNA - Chl), 9-16: pS002' (reporter - Chl) - pS004' seems to have worked beside C6
![](https://static.igem.org/mediawiki/2013/9/9f/13-09-30_colony_PCR_pS004%27_pS002%27.png)
13/09/30_Colony PCR_pS006 - didn’t work
![](https://static.igem.org/mediawiki/2013/b/bf/13-09-30_Colony_PCR_pS006.png)
other gel images that proof that the reporter as well as the Cas9 construct worked will follow soon!
Miniprep
pS013, pS004
1) Pellet liquid culture (4000rpm, 10 min)
2) Discard supernatant
3) resuspend the cells in 250ul of resuspension olution
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°
PCR: SPCR5, SPCR6 (gradient + touch down 55-75, -1°/cycle)
Reagent | Volume |
11x | |
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 |
Thermocycler Protocol: Fermentas Phusion | ||||
Temp | Time | |||
Start | 98°C | 30 sec | Melt | |
Cycle 1 | 98°C | 5 sec | Melt | 35 cycles |
Cycle 2 | 25 sec | Anneal | ||
Cycle 3 | 72°C | 30 sec per kb | Extend | |
Finish | 72 °C | 5 min | Extand | |
Store | 10°C | Forever | Store |
Liquid culture of pS004’ and pS013’