IGEM:Groningen/Notebook/iGEM 2011/2011/09/01

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1-9-11




Transformation of Berke's samples and RBS-GFP-DT from distribution kit
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


Colony PCR of cI-LVA in pSB1C3 and LasR-LVA in pSB1C3

Taq 10× buffer: 20μl
dNTPs 10mM: 4μl
MgCl2: 12μl
Forward Biobrick primer: 4μl
Reverse Biobrick primer: 4μl
Taq DNA polymerase: 1μl
MQ water: 155μl

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.

Cloning PBAD-A3DT and cI-LVA-A3DT
Calculations were made with the Ligation calculator based on DNA concentrations according to the nanodrop
Digestion:
PBADaraC
6μl insert (PBADaraC)
1μl EcoRI
1μl SpeI
2μl FD buffer
10μl MQ water

pSB1A3DTvector:
3μl vector
1μl EcoRI
1μl XbaI
3μl FD buffer
1μl FastAP
21μl MQ


cI-LVA
10μl insert
1μl XbaI
1μl PstI
2μl FD buffer
6μl MQ water

pSB1A3DTvector:
3μl vector
1μl XbaI
1μl PstI
3μl FD buffer
1μl FastAP
21μl 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
6μl PBADaraC
8.5μl vector RBS-GFP-DT
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
2.5μl MQ water

PBAD-RBS-GFP-DT
8.1μl RBS GFP ('insert')
8.5μl PBADaraCvector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
0.4μl MQ water

PBADaraC-cI-LVA
8.2μl cI-LVA
8.5μl PBADaraCvector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
0.3μl MQ water

PBADaraC-LasR-LVA
8.1μl LasR-LVA
8.5μl PBADaraCvector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
0.4μl 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

PCR to get cI-LVA and LasR-LVA with RBS
Per sample:
Pfu + MgSO4 buffer: 5μl
dNTPs 10mM: 1μl
Template: 1μl
Forward Biobrick primer: 1μl
Reverse Biobrick primer: 1μl
pfu DNA polymerase: 1μl
MQ water: 40μl

PCR conditions:
Preheated lid: 111°C
Denaturation: 94°C for 5 min.
Cycle 33×:
denaturation: 94°C for 30s.
annealing: 55,58(cI-LVA) and 60,63 (LasR-LVA)°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.
PCR product is OK, ND1000- Nanodrop shows: Concentration cI-LVA: 150 ng/μl and LasR-LVA is around 70ng/μl

Strains with promotor, RBS-cI-LVA-DT were grown for tomorrow's plasmid prep in LB with ampicillin. (distributed from iGEM
2008's team.





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