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Thursday 26^th 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.