Template:IONIS Paris/Notebook/25-05-15

RBS BioBricks

Recover dried DNA sent by iGEM (part BBa_J61100)

Plasmid characteristic:

  • Part name: BBa_J61100
  • Plasmid: pSB1A2
  • Part name: BBa_J61100
  • Resistance: AK (Amplicilin/Kanamicin)

Punch a hole without removing the foil
Pipette 10µl of water MQ (up and down)
Let sit for 5 minutes to make sure the dried DNA is fully resuspended
Recovered plasmid have been put into a 50µl eppendorf tube called “BBa_J61100, RBS”

Amplification of bFos, bJun, pSB1A2 (part BBa_J61100) into E.coli DH5 alpha

Plate preparation

Aim: preparation of selective LB agar plate

Add 40 ml of LB agar into a Falcon tube (50ml) + 40µl antibiotic For 20 ml / plate (2 plates)

Agar plates preparation
Plasmid Antibiotic Number of plate Remarks
bFos
Amplicillin
1
50 µl of transformed cells
1
100 µl of transformed cells
bJun
Amplicillin
1
50 µl of transformed cells
1
100 µl of transformed cells
pSB1A2
Ampicillin + Kanamycin
1
50 µl of transformed cells
1
100 µl of transformed cells
Control negative
Amplicilin
1
50 µl of transformed cells
Kanamycin
1
50 µl of transformed cells

Antibiotics concentration:

  • Ampicilin: 50 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)
1
bFos
1
2
bJun
1
3
pSB1A2
1
4
Control negative
--

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 2 control plates
Many colonies into all the other plates
Colonies from pSB1A2 transformation should exhibit red color


25 May 15