Template:IONIS Paris/Notebook/21-05-15

PCR VVD BioBricks

PCR.1 YFP Cter (YC155) & Nter (YN155)

Aim: amplification of YC155 and first step of mutagenesis for YN155

PCR YC155 and YN155 preparation
Component	PCR YC155 (x2)	PCR.1 YN155 I (x2)	PCR.1 YN155 II (x2)	Control - YC155	Control - YN155
Primer Fwd YC155	1 µl	--	--	--	--
Primer Rev YC155	1 µl	--	--	--	--
Primer Fwd YN155 I	--	1 µl	--	--	--
Primer Rev YN155 I	--	1 µl	--	--	--
Primer Fwd YN155 II	--	--	1 µl	--	--
Primer Rev YN155 II	--	--	1 µl	--	--
Plasmid pBiFC-bFos	1 µl	--	--	1 µl	--
Plasmid pBiFC-bJun	1 µl	--	--	1 µl	--
Taq Pol	0.5 µl	0.5 µl	0.5 µl	0.5 µl	0.5 µl

Mix dNTPs: 2 µl of each dNTP
Mix PCR without primers, plasmids and Taq Pol
PCR cycle IGEM TAQ program

Electrophoresis

Aim: check for digestion

1 gel: 120ml TAE 1X + 1,2g agarose + 150µl 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 of loading dye (6x) + 9 µl water MQ only 10 µl loaded.
110 V, 25 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 3 tubes: “PCR1, YC155, 21/05/2015”, “PCR1, YN155 I, 21/05/2015”, “PCR1, YN155 II, 21/05/2015”
Stored at -20°C.

Backbone & Terminator

Transformation pSB1C3 – double T7 terminator (BBa_B0015)

Aim: plasmid amplification

3 plates with 25 ml LB medium + 25 µl of Cam antibiotic / plate
Recover dried DNA sent by iGEM

Punch a hole without removing the foil (Plate 3, well F3)
Pipette 10 µl of water MQ (up and down)
Pipette 10 µl of water MQ (up and down)
Protocol for bacteria transformation

Cell transformation

E.coli DH5 alpha (NEB5 alpha competent coli)
Aliquot of 50 µl of competent E.coli / transformation

Into aliquots, added plasmids or nothing (control) as following:
Tube n°	Reagent added	Volume (µl)
1	pSB1C3 – double T7 terminator (BBa_B0015)	1
2	pSB1C3 – double T7 terminator (BBa_B0015)	4
3	Control negative	--

Incubation on ice, 30 min
Heat shock exactly 42°C, 30 seconds. Do not mix
Keep on ice for 5 min. Do not mix
400 µl of LB in sterile condition without antibiotic, pipette up and down gently
Incubation 37°C, 120 rpm, 1 hour
Centrifuge, 4000 rpm, 5 min
Remove and discard 300 µl of supernatant; resuspend gently using a pipette the remaining 100 µl
Plate bacteria and incubate at 37°C, O/N

Results
Control was cloudy as cultures (lack of antibiotics) should be done again
21 May 15