Team:Paris Bettencourt/Notebook/Phage Sensor/Monday 30th September.html

From 2013.igem.org

(Difference between revisions)
 
(One intermediate revision not shown)
Line 160: Line 160:
13/09/30_Colony PCR_pS006 - didn’t work<br>
13/09/30_Colony PCR_pS006 - didn’t work<br>
<img src="https://static.igem.org/mediawiki/2013/b/bf/13-09-30_Colony_PCR_pS006.png" width="400"/><br>
<img src="https://static.igem.org/mediawiki/2013/b/bf/13-09-30_Colony_PCR_pS006.png" width="400"/><br>
 +
<br>
 +
other gel images that proof that the reporter as well as the Cas9 construct worked will follow soon!
 +
<br>
<br>
<br>
<b>Miniprep</b><br>
<b>Miniprep</b><br>

Latest revision as of 00:59, 5 October 2013

Detect

Monday 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


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)


13/09/30_colony PCR_pS006'_pS006* - 1-8: pS006' (Cas9 - Chl), 9-16: pS006* (Cas9 - Amp) - didn’t work


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


13/09/30_colony PCR_pS004'_pS002' - 1-8: pS004' (gRNA - Chl), 9-16: pS002' (reporter - Chl) - pS004' seems to have worked beside C6


13/09/30_Colony PCR_pS006 - didn’t work


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’