Team:Paris Bettencourt/Notebook/Phage Sensor/Tuesday 17th September.html

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Revision as of 17:02, 25 September 2013

Phage Sensor

Tuesday 17th September

PCR Reaction (pS003 (Amp-G), pS004 (Amp-Lig), pS012 (Amp-G), pS013 (Chl-Lig))

PCR Reaction (pS003 (Amp-G), pS004 (Amp-Lig), pS012 (Amp-G), pS013 (Chl-Lig))

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
Forward Primer (10 uM) 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
Nuclease-free water 10 ul
Total Volume 25 ul



Thermocycler Protocol: NEB Quick-Load
Temp Time
Start 95°C 30 sec Melt
Cycle 1 95°C 15 sec Melt 35 cycles
Cycle 2 45.8 °C 30 sec Anneal
Cycle 3 72°C 1 min per kb Extend
Finish 72 °C 15 min Extand
Store 10°C Forever Store




Gels
100V, 30 min, 1%

Pictures: 13/09/17_colony PCR_pS009.JPG
13/09/17_colony PCR_pS008_pS002.jpg
13/09/17_colony PCR_pS003.JPG
13/09/17_colony PCR_pS004.JPG
13/09/17_colony PCR_pS012.JPG
13/09/17_colony PCR_pS013.JPG

Result:
pS003: row 3,5 might have worked -> analytical digest
pS004: all might have worked -> analytical digest
pS013: all might have worked -> analytical digest
pS012: row 3,4,5 might have workes -> analytical digest

Sequencing
Bringing primers/plasmids away for sequencin
Bacbbone, SPCR2 with iS002, iS032, iS028, iS029, iS030

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