IGEM:Groningen/Notebook/iGEM 2011/2011/09/06

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6-9-11



Plasmid Prep of PBAD-pSB1C3, PBAD-cI-LVA, PBAD-LasR-LVA and LasR-LVA in vector
Nanodrop ND1000 results: DNA in sample, between 30 and 60 ng/μl.

Colony PCR of PcI-RBS-GFP-DT and K077002

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-RBS-GFP-DT, PBAD-cI-LVA, PBAD-cI-LVA-DT and PBAD-LasR-LVA-DT
Calculations were made with the Ligation calculator based on DNA concentrations according to the nanodrop
Digestion:
PBADaraC
5μl insert (PBADaraC)
1μl EcoRI
1μl SpeI
2μl FD buffer
11μl MQ water

RBS-GFP-DTvector:
5μl vector
1μl EcoRI
1μl XbaI
3μl FD buffer
1μl FastAP
19μl MQ

cI-LVAvector:
5μl vector
1μl EcoRI
1μl XbaI
3μl FD buffer
1μl FastAP
19μl MQ

PBAD-PSB1C3vector:
5μl vector
1μl SpeI
1μl PstI
3μl FD buffer
1μl FastAP
19μl MQ

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

cI-LVA PCR product
3μl insert
1μl XbaI
1μl PstI
2μl FD buffer
13μl MQ water

PBAD-cI-LVA vector:
3μl vector
1μl SpeI
1μl PstI
3μl FD buffer
1μl FastAP
21μl MQ

PBAD-LasR-LVA vector:
5μl vector
1μl SpeI
1μl PstI
3μl FD buffer
1μl FastAP
19μl MQ

DT product
2μl insert
1μl XbaI
1μl PstI
2μl FD buffer
14μl MQ water

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
5.8μl PBADaraC
8.5μl vector RBS-GFP-DT
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
2.7μl MQ water

PBADaraC-cI-LVA
5.8μl PBADaraC
8.5μl vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
2.7μl MQ water

PBADaraC-RBS-GFP-DT
5.4μl RBS-GFP-DT
8.5μl vector PBAD-pSB1C3
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
3.1μl MQ water

PBADaraC-cI-LVA
4μl cI-LVA
8.5μl vector PBAD-pSB1C3
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
4.5μl MQ water

PBADaraC-cI-LVA-DT
2.3μl DT
8.5μl vector RBS-GFP-DT
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
6.2μl MQ water

PBADaraC-LasR-LVA-DT
2.3μl DT
8.5μl vector RBS-GFP-DT
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
6.2μl 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

Check PCR of autoinducing loop plasmids for Christoph and Vessa

Taq 10× buffer: 52μl
dNTPs 10mM: 10.4μl
MgCl2: 31.2μl
Forward Biobrick primer: 10.4μl
Reverse Biobrick primer: 10.4μl
Taq DNA polymerase: 2.6μl
MQ water: 403μl

PCR conditions:
Preheated lid: 111°C
Denaturation: 94°C for 10 min.
Cycle 30×:
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


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