Template:IONIS Paris/Notebook/07-05-15

PCR VVD BioBricks

PCR.1 VVD - 2nd part

Aim: first step for VVD amplification and mutagenesis

PCR.1 VVD preparation
Component	PCR.1 VVD 1 (x2)	PCR.1 VVD 2 (x2)
PCR mix	43,5 µl	43,5 µl
Primer VVD Fwd 1	1 µl	--
Primer VVD Rev 1	1 µl	--
Primer VVD Fwd 2	--	1 µl
Primer VVD Rev 2	--	1 µl
Water	2 µl	2 µl
Plasmid VVD	1 µl	1 µl
Taq Pol	0.5 µl	0.5 µl

Run of the IGEM TAQ program

Electrophoresis

Aim: check for DNA amplification

Low melting point gel
Sample: 10 µl of PCR product+ 2 µl of loading dye (6x)
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl of loading dye (6x) + 9 µl water MQ
110 V, 26 min

Expected results

Results

We get expected bands about 200 bp with a good intensity meaning DNA have been highly amplified without contamination.

Purification of PRC product

Aim: purify and concentrate amplified DNA

Use of a QIAquick PCR Purification kit and using a microcentrifuge.

For step 1: add 5 volumes of PB buffer for 1 volume of PCR product

200 µl of PB buffer added into both coupled of tube with 40 µl of remaining PCR product
Mix of same PCR product together into a new tube
Put mixes into their own purification column

Then, we followed the protocol from the supplier

At the end of the purification, we get 1 tube “PCR1, VVD1, 07/05/2015” and another one “PCR1, VVD2, 07/05/2015”
Stored at -20°C.

PCR.2 VVD

Aim: second step for VVD amplification and mutagenesis

dNTP preparation (4µL):
1 µl dATP 100 mM
1 µl dCTP 100 mM
1 µl dTTP 100 mM
1 µl dGTP 100 mM
Store at -20°C

PCR.2 VVD preparation
Component	PCR.2 VVD 1 (x2)	PCR.2 VVD 2 (x2)	Control negative (x2)
Primer VVD Fwd 1	1 µl	--	--
Primer VVD Rev 3	1 µl	--	--
Primer VVD Fwd 4	--	1 µl	--
Primer VVD Rev 2	--	1 µl	--
Water	39.5 µl	39.5 µl	41.5 µl
Buffer RB	5 µl	5 µl	5 µl
dNTPs	0.5 µl	0.5 µl	0.5 µl
Mg²⁺ 50mM	1.5 µl	1.5 µl	1.5 µl

7 May 15