Template:IONIS Paris/Notebook/04-09-15

Results of liquid cultures

After centrifugation and UV exposure, no green light into the pellet or the supernatant

Liquid culture on plates 96 wells of transformed bacteria with pDawn-toxine (Holin/Endolysin, CCDB, HOKD)

1 colonie / well with 200 µL of LB, 32 wells per type of transformation
37°C, 120 rpm without light exposure

  • DO595 to follow bacterial growth under no light exposure condition (no time to switch to light exposure condition and to follow the evolution of the DO595)

Digestion of miniprep from 03/09

Mix digestion preparation:
Tube Miniprep
H2O MQ 2 μL
Buffer 2.1 2 μL
DNA 10 μL
EcoRI 0,5 μL

37°C, 1h

Expected results

Expected results

Results

Results

Bands corresponding to pSB1C3-HOKD and pSB1C3-CCDB seem correct (PCR control required for checking)
About constructions with pDawn, bands at 2100 bp were not expected, and highest bands seem to get the same length require additional tests.

Ligation of pSB1C3 and pDawn Gblocks I-V

Mix ligation preparation:
Tube pDawn I-II-III
T4 ligase buffer 1,3 μL
pDawn I-V 12,2 μL
pSB1C3 0,5 μL
T4 ligase 0,5 μL

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

Transformation of 100 µL of competent cells; 30°C during the weekend

Digestion of miniprep from 03/09, pDawn-Gblocks

Mix digestion preparation:
Tube Miniprep
H2O MQ 13 μL
Buffer 2.1 2 μL
DNA 4 μL
EcoRI 0,5 μL
EcoRV 0,5 μL

37°C, 1h

Expected results

Expected results

Results

Results

No differences between the four digestion no parts into pDawn


4 September 15