IGEM:Groningen/Notebook/iGEM 2011/2011/08/25

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25-8-11



Transformation PBAD-cI-LVA and PBAD-LasR-LVA did not work: do it over again with recalculations.

Plasmid prep of the grown cultures from yesterday's transformants. According to the nanodrop: 50.5 and 70.3 nanogram/microliter
DNA in sample. Glycerol stock was made. Use plasmid prep sample for digestion.

Digestion:

PBAD-pSB1C3
4μl vector
1μl SpeI
1μl PstI
3μl FD buffer
1μl FastAP
20μl MQ

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

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

PhybB-cI-LVA vector
3.5μl vector
1μl SpeI
1μl PstI
3μl FD buffer
1μl FastAP
20.5μl MQ water

PhybB-LasR-LVA vector
2.5μl vector
1μl SpeI
1μl PstI
3μl FD buffer
1μl FastAP
21.5μl MQ water

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

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

Ligation:

PBAD-cI-LVA
8.5μl vector
6.4μl insert
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
2.1μl MQ water

PBAD-LasR-LVA
8.5μl vector
7.5μl insert
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
1μl MQ water

PhybB-cI-LVA-DT
8.5μl vector
6μl insert
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
2.5μl MQ water

PhybB-LasR-LVA
8.5μl vector
6μl insert
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
2.5μl MQ water

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

Transformation of overnight ligation of PBAD-RBS-cI-LVA and PBAD-RBS-LasR-LVA
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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