From 2007.igem.org

PCRs

• The assembly PCRs did not work... we'll try them again !
• The Lox66-DapAColi PCRs worked very well in all conditions

=> Gel purification

PCR : FtsZ-E.Coli
Name	Ftsz-E.Coli
Annealing T°	55°C
Time of elongation	2m30'
Number of Cycles	30
Buffer	5x 10µL
MgCl2	10µM 0µL
dNTP	10µM 1µL
oligoF	1 Lox71-FtsZ-F	10µM 2.5µL
oligoR	2 FtsZ-R	10µM 2.5µL
water	34µL
polymerase	Phusion 0.5µL
DNA	toothpick in glycerol stock of MG1655

PCR : DapA-Subtilis
Name	DapA-Subtilis
Annealing T°	55°C
Time of elongation	2m30'
Number of Cycles	30
Buffer	5x 10µL
MgCl2	10µM 0µL
dNTP	10µM 1µL
oligoF	8 Lox66-DapASubtilis-F	10µM 2.5µL
oligoR	9 DapASubtilis R	10µM 2.5µL
water	34µL
polymerase	Phusion 0.5µL
DNA	0.5µL of Subtilis DNA from stock (DB tube in fridge)

PCR : Assembling PCR DGAT
Name	assembly PCR of DGAT
Annealing T°	55°C
Time of elongation	2m30'
Number of Cycles	30
Buffer	5x 10µL
MgCl2	10µM 0µL
dNTP	10µM 1µL
oligoF		10µM 2.5µL
oligoR		10µM 2.5µL
water	34µL
polymerase	Phusion 0.5µL
DNA	1µL of PCR product DGAT 1 1µL of PCR product DGAT 2

PCR : Assembling PCR FtsZ-FtsA
Name	assembly PCR of FtsZ-FstA
Annealing T°	55°C
Time of elongation	2m30'
Number of Cycles	30
Buffer	5x 10µL
MgCl2	10µM 0µL
dNTP	10µM 1µL
oligoF	3	10µM 2.5µL
oligoR	2	10µM 2.5µL
water	34µL
polymerase	Phusion 0.5µL
DNA	1µL of PCR product FtsZ 1 1µL of PCR product FtsZ 2

DAP solution contamination test

We spread on LB plates DAP solution :

• 50µL of aliquot DAP 50mM (in use)
• 50µL of DAP 1mM
• 50µL of DAP 0.2 mM
• 50µL of DAP 50mM