Template:IONIS Paris/Notebook/14-09-15

PCR Nanoluc

Mix PCR preparation:
Tube pSB1C3-Gblock
MQ Water 40 µL
RB Buffer 5 µL
Mg2+ 1,5 µL
dNTP 10 µM 1 µL
Primer Fwd 0,5 µL
Primer Rev 0,5 µL
DNA 1µL
Taq Pol 0,5 µL
PCR cycle – IGEM TAQ NanoLuc
T°C Time Cycle
Initial denaturation
98
5 min
1
Denaturation
98
30 sec
17
Annealing
58
30 sec
Extension
72
1 min
Final extension
72
5 min
1
Hold
4
Infinite

PCR colony for pSB1C3-VVD YC + YN and pSB1C3-VVD YN + YC

Mix PCR preparation:
Tube pSB1C3-VVD YC + YN (x7) pSB1C3-VVD YN + YC (x6)
MQ Water 40 µL 40 µL
RB Buffer 5 µL 5 µL
Mg2+ 1,5 µL 1,5 µL
dNTP 10 µM 1 µL 1 µL
Primer Fwd YC Fwd, 0,5 µL pSB1C3 Fwd 2, 1 µL
Primer Rev pSB1C3 Rev 2, 1 µL YN Rev 1, 0,5 µL
Taq Pol 0,5 µL 0,5 µL
PCR cycle – IGEM TAQ
T°C Time Cycle
Initial denaturation
98
5 min
1
Denaturation
98
30 sec
35
Annealing
51
30 sec
Extension
72
2 min
Final extension
72
5 min
1
Hold
4
Infinite
Expected results

Expected results

Results

Results

Not expected bands for every PCR colony Could be due to the choice of primers

  • Make another one with primers for pSB1C3 (more simple)

Digestion

Mix digestion preparation:
Tube pSB1C3-pDawn pSB1C3 iGEM Nanoluc
H2O MQ - - 14 μL
Buffer 2.1 1,2 μL 1,1 μL 2 μL
DNA 10 μL 8 μL 2 μL
Enzyme 1 XbaI 1 μL EcoRI 1 μL EcoRI 1 μL
Enzyme 2 - PstI 1 μL PstI 1 μL

37°C, 1h heat kill: 80°C, 20min

Ligation

Mix ligation preparation:
Tube pSB1C3-pDawn pSB1C3-pDawn I-V (1:2) pSB1C3-pDawn I-V (2:1) pSB1C3-nanoluc (1:1) pSB1C3-nanoluc (1:2)
H2O MQ - - - 0,8 μL 4,3 μL
T4 buffer 1,4 μL - 0,5 μL 0,5 μL 1 μL
pSB1C3 - 1,4 µL 5,5 µL 7 µL 3,5 µL
Insert 12,2 μL 20 μL 22 μL 0,7 μL 0,7 μL
T4 ligase 0,5 μL 1 μL 1 μL 0,5 μL 0,5 μL

Room temperature, 45min heat kill: 65°C, 20min

  • Transformation of competent cells with all ligation products using 1 µL or 3 µL

PCR colony for pSB1C3-VVD YC + YN and pSB1C3-VVD YN + YC

Mix PCR preparation:
Tube pSB1C3-VVD YC + YN (x7) pSB1C3-VVD YN + YC (x7)
MQ Water 40 µL 40 µL
RB Buffer 5 µL 5 µL
Mg2+ 1,5 µL 1,5 µL
dNTP 10 µM 1 µL 1 µL
Primer pSB1C3 Fwd 1 µL 1 µL
Primer pSB1C3 Rev 1 µL 1 µL
Taq Pol 0,5 µL 0,5 µL
Expected results

Expected results

Results

Results

No differences with previous PCR colony
Bands weight do not correspond to the two possible cases “no insertion” or “insertion” of the part

  • Liquid culture of 1 colony of each to analyse the DNA

Bacterial growth kinetic of bacteria transformed with pDawn-H/E

Liquid culture of 100 ml LB + 100 µl Kan for 5 interesting colonies from the experiment of the 09/09
37°C, 120 rpm, O/N, no light exposition


14 Sept. 15