Team:Groningen/project notebook/2 September 2011

Olesja
Flowcytometry measurement (promoter leakage)
constructs:

• Prm_GFP_DT
• PLasR_GFP_DT
  

Joyce

Plasmid Prep of overnight cultures: cI-LVA and LasR-LVA in chloramphenicol vector+ K077002 and K07004 for cI production.
According to the ND1000- Nanodrop, DNA concentration was: around 30ng/μl

Use the cI-LVA and LasR-LVA vectors for digestion

Digestion:

PBAD
5.6 μl PBAD PCR product
1μl EcoRI
1μl SpeI
2μl FD buffer
10.4μl MQ water

cI-LVA vector:
4μl cI-LVA vector
1μl EcoRI
1μl XbaI
3μl FD buffer
1μl FastAP
20μl MQ water

LasR-LVA vector:
4μl LasR-LVA vector
1μl EcoRI
1μl XbaI
3μl FD buffer
1μl FastAP
20μl MQ water

pSB1C3-DT vector:
3μl pSB1C3-DT
1μl EcoRI
1μl XbaI
3μl FD buffer
1μl FastAP
21μl MQ water

pSB1C3-DT vector
3μl pSB1C3-DT
1μl XbaI
1μl PstI
3μl FD buffer
1μl FastAP
21μl MQ water

LasR-LVA PCR product:
4μl LasR-LVA PCR product
1μl XbaI
1μl PstI
2μl FD buffer
12μl MQ water

Incubate the samples for 1h at 37°C

After digestion: DNA clean up with the High Pure PCR Purification kit of Roche

Ligation:

PBAD-cI-LVA
5μl PBAD
8.5μl cI-LVA vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
3.5μl MQ water

PBAD-LasR-LVA
5μl PBAD
8.5μl LasR-LVA vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
3.5μl MQ water

PBAD-pSB1C3DT
7μl PBAD
8.5μl pSB1C3DT vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
1.5μl MQ water

LasR-LVA in pSB1C3DT vector
8.5μl LasR-LVA PCR product
8.5μl pSB1C3DT vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase

Ligate for 30-40 minutes at room temperature

Transformation:
Add to 40μl competent E.coli DH5alha cells 10μl ligation mixture
Incubate the samples for 30 min. on ice
Heat shock:
Incubate the samples for 45s at 42°C
After the heat shock: place the cells for 2 min on ice and add after the incubation time on ice 1ml of LB+25mM glucose.
Incubate the samples for 1h to 1.5 h at 37°C
Plate the cells out: Centrifuge the sample, leave 100μl of LB+glucose in the tube and resuspend the pellet.
Plate 90μl and 10μl of the resuspended pellet on plates containing antibiotic(s)

Colony PCR
Colony PCR of the RBS-GFP-DT plasmid from the distribution kit. Yesterday, that sample had been transformed again, since there
were some difficulties with the plasmid in cloning purposes. It did not seem to have the insert (RBS-GFP-DT) anymore.

PCR mix:
10× Taq buffer: 20μl
dNTPs 10mM each: 4μl
MgCl2: 12μl
BB forward primer: 4μl
BB reverse primer: 4μl
Taq polymerase: 1μl
MQ water: 155μl

PCR conditions:
Pre heated lid at 111°C
Denaturation: 94°C for 10 min
Cycle 33×
Denaturation: 94°C for 30s
Annealing: 60°C for 30s
Extension: 72°C for 2.5 min
Final extension: 72°C for 10 min
Store at 4°C infinite

Analyse on a 1% TBE agarose gel