IGEM:Groningen/Notebook/iGEM 2011/2011/09/05

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5-9-11



Colony PCR of PBAD-cI-LVA in pSB1C3, PBAD-LasR-LVA in pSB1C3, PBAD in pSB1C3 and LasR-LVA in pSB1C3. Also a PCR for obtaining
PCR product of RBS-GFP-DT

Taq 10× buffer: 76μl
dNTPs 10mM: 15.2μl
MgCl2: 45.6μl
Forward Biobrick primer: 15.2μl
Reverse Biobrick primer: 15.2μl
Taq DNA polymerase: 3.8μl
MQ water: 589μl

For RBS-GFP-DT PCR product per sample:
Pfu buffer+ MgSO4 10× buffer: 5μl
dNTPs 10mM: 1μl
Forward Biobrick primer: 1μl
Reverse Biobrick primer: 1μl
Pfu polymerase: 1μl
MQ water: 40μl

PCR conditions:
Preheated lid: 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 infinite at 4°C

Analyse on a 1% TBE agarose gel.

Plasmid prep of RBS-GFP-DT vector. Glycerol stock had been made and plasmids were purified. Although, the centrifuge went down,
so the samples stood longer than is normally required. After measurements with the ND-1000 Nanodrop, the DNA concentration was
little lower than usual: 30.8 ng/μL and 10.5ng/μL.

Cloning PBAD-RBS-GFP
Calculations were made with the Ligation calculator based on DNA concentrations according to the nanodrop
Digestion:
RBS GFP from plasmid
16μl insert (PBADaraC)
1μl XbaI
1μl PstI
2μl FD buffer

Digestion:
RBS GFP PCR product
5μl insert (PBADaraC)
1μl XbaI
1μl PstI
2μl FD buffer
11μl MQ water

PBAD-pSB1C3vector:
4μl vector
1μl XbaI
1μl PstI
3μl FD buffer
1μl FastAP
20μl MQ

Incubate the samples for 1h at 37 °C.

DNA clean up with the High Pure PCR Purification Kit of Roche

Ligation:
PBADaraC-RBS-GFP-DT (insert from plasmid)
7.4μl PBADaraC
8.5μl vector RBS-GFP-DT
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
1.1μl MQ water

PBADaraC-RBS-GFP-DT (insert from PCR product)
5.6μl PBADaraC
8.5μl vector RBS-GFP-DT
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
2.9μl MQ water

Self ligation control:
8.5μl vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
8.5μl MQ water

Incubate the samples for 30 to 40 minutes at roomtemperature

Transformation
Add to 40μl competent cells 10μl ligation mixture
Incubate for 30 min on ice
Heat shock: 45s at 42 degrees
Incubate cells on ice for 2 min
Add 1ml LB medium+ 25mM glucose
Incubate cells for 1h/1.5h at 37 degrees
Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates
Put the plates in the stove at 37 degrees overnight

Grow colonies: PBAD-LasR-LVA and PBAD-pSB1A3DT and LasR-LVA in vector






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