Template:IONIS Paris/Notebook/22-05-15

PCR VVD BioBricks

PCR.2 Nter (YN155)

Aim: second step of mutagenesis for YN155

PCR.2 YN155 preparation

Component	PCR.2 YN155 I (x2)	PCR.2 YN155 II (x2)	Control - YN155 I (x2)	Control - YN155 II
Primer Fwd YN155 1	1 µl	--	--	--
Primer Rev YN155 3	1 µl	--	--	--
Primer Fwd YN155 4	--	1 µl	--	--
Primer Rev YN155 2	--	1 µl	--	--
PCR.1, YN155 I	1 µl	--	1 µl	--
PCR.1, YN155 II	--	1 µl	--	1 µl
Taq Pol	0.5 µl	0.5 µl	0.5 µl	0.5 µl

Mix PCR (x8) without primers, DNA and Taq pol
PCR cycle IGEM TAQ program, Tm = 61,3°C

Electrophoresis

Aim: check for digestion

Remaining gel (120ml TAE 1X + 1,2g agarose + 150µl BET) with 8 wells
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 of loading dye (6x) + 9 µl water MQ only 10 µl loaded.
110 V, 26 min

Expected results

Results

We get expected bands with a good intensity 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 2 tubes: “PCR2, YN I, 22/05/2015”, “PCR2, YN II, 22/05/2015”
Stored at -20°C.

Liquid culture

Use of 3 colonies (3 tubes) from pSB1C3 (BBA_B0015) 1µl and pSB1C3 (BBA_B0015) 4µl
3ml LB Broth + 3µl Cam in double click tubes (12ml) + 1 control tube (3ml LB Broth)
37°C, 120rpm, O/N

22 May 15