Template:IONIS Paris/Notebook/13-05-15

PCR VVD BioBricks

PCR.2 VVD - 2nd part

Aim: second step for VVD amplification and mutagenesis

End of PCR.2 VVD preparation from 07.05.15

Component	PCR.2 VVD 1 (x2)	PCR.2 VVD 2 (x2)	Control negative	Control negative
DNA Mix "PCR.1 VVD1"	1 µl	--	1 µl	--
DNA Mix "PCR.1 VVD2"	--	1 µl	--	1 µl
Taq Pol	0.5 µl	0.5 µl	0.5 µl	0.5 µl

Run of the IGEM TAQ program

Electrophoresis

Aim: check for digestion

1 gel: 120ml TAE 1X + 1,2g agarose + 160µl BET
Sample: 10 µl of PCR product+ 2 µl of loading dye (6x) only 10 µl loaded.
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl loading dye (6x) + 9 µl water MQ only 10 µl loaded.
110 V, 30 min

Expected results

Results

We get expected bands about 250 bp meaning DNA have been highly amplified

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 “PCR2, VVD1, 13/05/2015” and another one “PCR2, VVD2, 13/05/2015”
Stored at -20°C.

13 May 15