Template:IONIS Paris/Notebook/30-06-15

PCR pDawn BioBricks

PCR YC155, pDawn I, II and III

Aim: first step for pDawn amplification and mutagenesis

Mix PCR preparation:

	YC155	pDawn I	pDawn II	pDawn III
MQ water	39,5 µL	39,5 µL	39,5 µL	39,5 µL
Mg2+ 50 mM	1,5 µL	1,5 µL	1,5 µL	1,5 µL
Buffer RB	5 µL	5 µL	5 µL	5 µL
dNTPs 10µL	1 µL	1 µL	1 µL	1 µL
Primer Fwd 50µM	1µL YC155 Fwd	1µL pDawn Fwd I	1µL pDawn Fwd II	1 µL pDawn Fwd III
Primer Rev 50µM	1µL YC155 Rev	1µL pDawn Rev I	1µL pDawn Rev II	1 µL pDawn Rev III
DNA	1µL PCR YC155	1µL pDawn	1µL pDawn	1 µL pDawn
Taq Pol	0.5 µL	0.5 µL	0.5 µL	0.5 µL

iGEMTAQ program

Electrophoresis

Aim: Migration of PCR product

Gel : 1,2 g agarose + 120 ml of TAE 1x + 150 µl of BET
Migration of PCR products YC155, pDawn I, II and III

Expected results

Results

We get expected bands for YC155, pDawn I and II but there is no amplification for pDawn III
First explanation: human error We should make the reaction again

Gel Purification

Aim: purification of fragment from PCR

QIAquick gel extraction
2 bands of each fragment into 1 column
Concentrate into 50 µl of Elution Buffer

PCR YC155, pDawn I, II and III

Mix PCR preparation:

	pDawn III
MQ water	39,5 µL
Mg2+ 50 mM	1,5 µL
Buffer RB	5 µL
dNTPs 10µL	1 µL
Primer Fwd 50µM	1µL pDawn Fwd III
Primer Rev 50µM	1µL pDawn Rev III
DNA	1µL pDawn
Taq Pol	0.5 µL

iGEMTAQ program

Electrophoresis

Aim: Migration of PCR product

Gel : 1,2 g agarose + 120 ml of TAE 1x + 150 µl of BET
Migration of PCR products purified YC155 and pDawn III

Expected results

Results

No amplification of pDawn III
YC155 quantification = 128 ng.µL^-1

30 June 15