Template:IONIS Paris/Notebook/10-07-15

PCR pDawn BioBricks

PCR 2 pDawn I,II and III

Aim: second step for pDawn amplification and mutagenesis
Mix PCR preparation:
pDawn I pDawn II pDawn III
MQ water 39,5 µL 39,5 µL 39,5 µL
Mg2+ 50 mM 1,5 µL 1,5 µL 1,5 µL
Buffer RB 5 µL 5 µL 5 µL
dNTPs 10µM 1 µL 1 µL 1 µL
Primer Fwd 50µM 1µL pDawn Fwd I 1µL pDawn Fwd V 1µL pDawn Fwd VI
Primer Rev 50µM 1µL pDawn Rev IV 1µL pDawn Rev V 1µL pDawn Rev VI
DNA 1µL pDawnI PCR1 1µL pDawnII PCR1 1µL pDawnIII PCR1
Taq Pol 0.5 µL 0.5 µL 0.5 µL
PCR cycle – IGEM TAQ 2
T°C Time Cycle
Initial denaturation
95
30 sec
1
Denaturation
95
30 sec
17
Annealing
60
30 sec
Extension
72
2 min
Final extension
72
5 min
1
Hold
4
Infinite

Electrophoresis


Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of pDawn I, II and III

Expected results

Expected results

Results

Results

Good amplification of pDawnI and II
The amplification of pDawn 3 is unexpected and cannot be used for the following experiment
About the miniprep of bacteria transformed with the Gibson assembly product: two plasmids have been purified (only one expected); additional checking steps will be lead to characterize these plasmids

Gel purification


QIAquick gel extraction
2 bands of each fragment (pDawnI and II) into 1 column
Concentrate into 30 µl of Elution Buffer

PCR amplification of pDawnIII

Aim: step for pDawn amplification and mutagenesis
Mix PCR preparation:
pDawn III pDawn III
MQ water 35,5 µL 25,5 µL
High GC content Buffer / 10 µL
Buffer Q5 10 µL 10 µL
dNTPs 10µM 1 µL 1 µL
Primer Fwd 50µM 1µL pDawn Fwd III 1µL pDawn Fwd III
Primer Rev 50µM 1µL pDawn Rev III 1µL pDawn Rev III
DNA 1µL pDawnIII PCR1 1µL pDawnIII PCR1
Taq Pol 0.5 µL 0.5 µL
PCR cycle – IGEM TAQ 3
T°C Time Cycle
Initial denaturation
98
30 sec
1
Denaturation
98
30 sec
17
Annealing
60
30 sec
Extension
72
2 min
Final extension
72
5 min
1
Hold
4
Infinite

Electrophoresis


Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of pDawn III

Expected results

Expected results

Results

Results

Small amplification of pDawnIII

Gel purification


QIAquick gel extraction
4 bands of pDawnIII into 4 columns (due to high volume of buffer)
Concentrate into 25 µl x4 of Elution Buffer

Production of competent cells (3rd step)


Massive liquid culture into an Erlenmeyer of 500 ml (100 ml of LB medium) using few ml of pre-liquid culture
Application of the protocol for the production of competent cells 95 aliquots of 100µL


10 July 15