IGEM:Groningen/Notebook/iGEM 2011/2011/08/29

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29-8-11


Cloning PBAD-RBS-GFP (in 2 different ways), PBAD-cI-LVA and PBAD-LasR-LVA
Calculations were made with the Ligation calculator based on DNA concentrations according to the nanodrop
Digestion:
PBADaraC
4μl insert (PBADaraC)
1μl EcoRI
1μl SpeI
2μl FD buffer
12μl MQ water

RBS-GFP-DT vector:
3μl vector
1μl EcoRI
1μl XbaI
3μl FD buffer
1μl FastAP
21μl MQ

RBS-GFP-DT
14μl ´insert´
1μl XbaI
1μl PstI
2μl FD buffer
2μl MQ water

PBADaraC vector
4μl vector
1μl SpeI
1μl PstI
3μl FD buffer
1μl FastAP
20μl MQ water

cI-LVA
7μl insert
1μl XbaI
1μl PstI
2μl FD buffer
9μl MQ water

LasR-LVA
12μl insert
1μl XbaI
1μl PstI
2μl FD buffer
4μl 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
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


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