Template:IONIS Paris/Notebook/08-09-15

Digestion

Mix digestion preparation:
Tube VVD-YC155 VVD-YN155
Buffer 2.1 1.4 µL 1.4 µL
DNA 12 µL 12 µL
Enzyme 1 (0,5 µL) EcoRI XbaI
Enzyme 2 (0,5 µL) SpeI PstI
Mix digestion preparation:
Tube pSB1C3 GFP Holin/Endolysin
Buffer 2.1 1 µL 1,4 µL 1,4 µL
DNA 8 µL 12 µL 12 µL
EcoRI 0,5 µL 0,5 µL 0,5 µL
PstI 0,5 µL 0,5 µL 0,5 µL

37°C, 1h heat kill: 80°C, 20min

Ligation

Mix ligation preparation:
Tube pSB1C3-VVD YC-VVD YN
H2O MQ 0,2 μL
T4 ligase buffer 1 μL
VVD-YC 2 μL
VVD-YN 2,6 μL
pSB1C3 3,7 μL
T4 ligase 0,5 μL

Room temperature, 30 min heat kill: 65°C, 20min

  • Transformation of bacteria with 1 µL or 4 µL of pSB1C3-VVD YC-VVD YN

Mix ligation preparation:
Tube pSB1C3-GFP pSB1C3-H/E
Ratio (backbone:insert)
3:1
1:1
1:3
3:1
1:1
1:3
H2O MQ 6,6 μL 5,7 μL 3 μL 6,4 μL 5 μL 0,7 μL
T4 ligase buffer 1 μL 1 μL 1 μL 1 μL 1 μL 1 μL
Insert 0,5 μL 1,4 μL 4,1 μL 0,7 μL 2,1 μL 6,4 μL
pSB1C3 1,4 μL 1,4 μL 1,4 μL 1,4 μL 1,4 μL 1,4 μL
T4 ligase 0,5 μL 0,5 μL 0,5 μL 0,5 μL 0,5 μL 0,5 μL

Room temperature, 30min heat kill: 65°C, 20min

PCR pSB1C3-Gblock (CCDB, HOKD, VVD YC, VVD YN)

Mix PCR preparation:
Tube pSB1C3-Gblock
MQ Water 40 µL
RB Buffer 5 µL
Mg2+ 1,5 µL
dNTP 10 µM 1 µL
Primer pSB1C3 Fwd 1 µL
Primer pSB1C3 Rev 1 µL
DNA 1µL
Taq Pol 0,5 µL

Run of the IGEM TAQ program

Cultures from 07/09

Many colonies for pDawn-H/E, pDawn-CCDB, pDawn-HOKD and pDawn-GFP
Few colonies for pSB1C3-pDawn I-V

PCR colonies for pSB1C3-pDawn I-V

Mix PCR preparation:
Tube pSB1C3-pDawn I-V
MQ Water 40 µL
RB Buffer 5 µL
Mg2+ 1,5 µL
dNTP 10 µM 1 µL
Primer pDawn Fwd 2 0,8 µL
Primer pDawn Rev 2 0,8 µL
Taq Pol 0,5 µL

Liquid culture

Liquid culture on 96 wells plates for pDawn-H/E, pDawn-CCDB, pDawn-HOKD and pDawn-GFP:

  • 1st plate (for each plasmid) has filled with 250 µL of LB + 25 µL of Kanamycin, 1 well on 2.1 colony / well
    Withdraw 125 µL/well and fill the same well of a 2nd plate (save)
    Measure the DO595 of 1st plates and let it at 37°C, 120 rpm, O/N with light exposition


8 September 15