Template:IONIS Paris/Notebook/25-08-15

Results of liquid cultures

No bacteria into the control, many bacteria everywhere else
Miniprep (QIAgen)

Digestion of pSB1C3 (from iGEM), miniprep and Gblocks

Miniprep for pDawn and VVD-YN155
Gblocks: ordered fragment from IDT (VVD-YC155, VVD-YN155, GFP rev, Holin/Endolysin rev (H/E rev), CCDB rev, HOKD rev)

Mix digestion preparation:
Tube pSB1C3 Miniprep Gblocks
Water 6 µL 12 µL 11 µL
Buffer 2.1 4 µL 2 µL 2 µL
Plasmid 8 µL 5 µL 6 µL
Enzyme EcoRI 1 µL 0,5 µL 0,5 µL
Enzyme PstI 1 µL 0,5 µL 0,5 µL

37°C, 1h heat kill: 80°C, 20min

PCR biobrick pSB1C3_VVD-YC155

Mix PCR preparation:
VVD-YC155
MQ Water 40 µL
RB Buffer 5 µL
Mg2+ 1,5 µL
dNTP 10 µM 1 µL
Primer Fwd 50 µM 0,5µl VVD Fwd I
Primer Rev 50 µM 1µl pSB1C3_VVD-YC155
Q5 enzyme 0,5 µL
PCR cycle – IGEM TAQ
T°C Time Cycle
Initial denaturation
98
5 min
1
Denaturation
98
30 sec
17
Annealing
60
30 sec
Extension
72
2 min
Final extension
72
10 min
1
Hold
4
Infinite
Expected results

Expected results

Results

Results

Amplification of VVD-YC155 validation of the biobrick
VVD-YN: bands at 2000 bp only or 2000 bp and 1000 bp (profile of pSB1C3 with RFP) no part
pDawn: bands at 2000 bp only or 2000 bp and 1000 bp (profile of pSB1C3 with RFP) no part

Ligation of Gblocks into pSB1C3

Mix ligation preparation:
Tube VVD-YC155 VVD-YN155 GFP rev H/E rev CCDB rev HOKD rev
Water 9,5 µL 11,2 µL 9,5 µL 12,3 µL 7 µL 4,5 µL
T4 ligase Buffer 2 µL 2 µL 2 µL 2 µL 2 µL 2 µL
Gblock 5 µL 3,3 µL 5 µL 2,2 µL 7,5 µL 10 µL
pSB1C3 2,5 µL 2,5 µL 2,5 µL 2,5 µL 2,5 µL 2,5 µL
T4 ligase 1 µL 1 µL 1 µL 1 µL 1 µL 1 µL

Room temperature, 30min

Expected results

Expected results

Results

Results

Ligation: epic fail


25 August 15