Template:IONIS Paris/Notebook/30-03-15

Amplification of pDusk, pDawn, pSB1C3, VVD, pUC19 into E.coli DH5 alpha

Plate preparation

Aim: preparation of selective LB agar plate

Add 50 ml of LB agar into a Falcon tube (50ml) + 50 µl antibiotic
For 25 ml / plate (2 plates)

Agar plates preparation
Plasmid Antibiotic Number of plate Remarks
pDusk
Kanamycin
1
50 µl of transformed cells
1
100 µl of transformed cells
1
50 µl of competent cells (neg control)
pDawn
Kanamycin
1
50 µl of transformed cells
1
100 µl of transformed cells
1
50 µl of competent cells (neg control)
pSB1C3
Chloramphenicol
1
50 µl of transformed cells
1
100 µl of transformed cells
1
50 µl of competent cells (neg control)
VVD
Amplicilin
1
50 µl of transformed cells
1
100 µl of transformed cells
1
50 µl of competent cells (neg control)
pUC19 Amplicilin
1
50 µl of transformed cells

Summary:
6 plates with Kanamycin
3 plates with Chloramphenicol
4 plates with Ampicillin

Antibiotics concentration:
Ampicillin: 50 µg.ml-1 diluted 1/1000
Chloramphenicol: 25 µg.ml-1 diluted 1/1000
Kanamycin: 50 µg.ml-1 diluted 1/1000


Cell transformation

E.coli DH5 alpha (NEB5 alpha competent coli)
Stock: 6 tubes 0.20 ml / tube; 50 pg.µl-1
Aliquot of 50 µl of competent E.coli / transformation

Into aliquots, added plasmids or nothing (control) as following:
Tube n° Reagent added Volume (µl) Tube n° Reagent added Volume (µl)
1
pDusk
1
5
pSB1C3
1
2
pDawn
1
6
(Amp control)
/
3
VVD
1
7
(Kan control)
/
4
pUC19
1
8
(Cam control)
/

Incubation on ice, 30 min
Heat shock exactly 42°C, 30 seconds. Do not mix
Keep on ice for 5 min, do not mix
400 µl of LB in sterile condition without antibiotic, pipette up and down gently
Incubation 37°C, 120 rpm, 1 hour
Centrifuge, 4000 rpm, 5 min
Remove and discard 300 µl of supernatant; resuspend gently using a pipette the remaining 150 µl
Plate bacteria following the table about plate preparation
Incubate at 37°C, O/N

Expected results:
No colonies into the 3 control plates
Many colonies into all the other plates;
Colonies from pSB1C3 transformation should exhibit red color


30 March 15