Team:Paris Bettencourt/Notebook/Phage Sensor/Thursday 26th September.html

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Detect

Thursday 26th September

Gel PCR 5,6, Digestion, PCR (SPCR7-10) and PCR purification

Gel PCR 5,6 1%, 100V, 1h

Picture: 13/09/26_SPCR5_6 - PCR didn't work


Digestions: SPCR2 (EcoRI, PstI), SPCR3 (EcoRI, PstI), SPCR4 (EcoRI, PstI), SPCR11 (EcoRI, PstI), lin pSB1C3 (EcoRI, PstI), lin pSB1A3 (EcoRI, PstI), SPCR5 (XbaI, SpeI), SPCR7 (XbaI, SpeI), lin pSB1C3 (XbaI, SpeI), in pSB1A3 (XbaI, SpeI)

Reagent Volume
Purified PCR Product 16 µl
H2O 0 µl
10x FastDigest Buffer 2 µl
FastDigest Enzyme 1 1 µl
FastDigest Enzyme 2 1 µl
Total Volume 20 µl


PCR (SPCR7-10)

Reagent Volume
x1
Nuclease-free water 37.25 µl
5x Phusion HF Buffer 10 µl
10 mM dNTPs 1 µl
Forward Primer (10 uM) 1 µl
Reverse Primer (10 uM) 1 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



PCR Purification: (SPCR2 (EcoRI, PstI), SPCR3 (EcoRI, PstI), SPCR4 (EcoRI, PstI), SPCR11 (EcoRI, PstI), lin pSB1C3 (EcoRI, PstI), lin pSB1A3 (EcoRI, PstI), SPCR5 (XbaI, SpeI), SPCR7 (XbaI, SpeI), lin pSB1C3 (XbaI, SpeI), in pSB1A3 (XbaI, SpeI)

Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through. Place the QIAquick column back into the same tube
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 50 μl Buffer EB to the center of the QIAquick membrane and centrifuge the column for 1 min.