IGEM:Groningen/Notebook/iGEM 2011/2011/08/31

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31-8-11



Making schemes for this week: Cloning strategy
Today clone cI-LVA and LasR-LVA in pSB1C3-DT, but digest with X and P because the PCR products only contain the XbaI site.
This means that the DT will be excluded. This also means that an extra cloning step will be included to clone the double
terminator in the vector.
Tomorrow: Colony PCR of transformants, also clone PBAD vector in pSB1A3DT.
Friday: Colony PCR of PBAD in pSB1A3DT and if there were right transformants yesterday: plasmid prep, digestion cI-LVA and
LasR-LVA in vector and digest the double terminator PCR product, clean up, ligation, transformation.

So today:
Digestion
pSB1C3DT
3μl vector
1μl XbaI
1μl PstI
3μl FD buffer
1μl FastAP
21μl MQ water

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

LasR-LVA
11μl
1μl XbaI
1μl PstI
2μl FD buffer
5μl MQ water

Incubate the samples at 37°C for 1h

Clean up the DNA with the High Pure PCR purification kit

Ligation:

cI-LVA-pSB1C
8.5μl vector pSB1C3-DT
8.5μl insert
2μl T4DNA ligase buffer
1μl T4 DNA ligase

LasR-LVA-pSB1C
8.5μl vector pSB1C3-DT
8.5μl insert
2μl T4DNA ligase buffer
1μl T4 DNA ligase

Self ligation pSB1C3-DT
8.5μl vector pSB1C3-DT
8.5μl MQ water
2μl T4DNA ligase buffer
1μl T4 DNA ligase

Incubate samples for 35 min. at room temperature

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

At 14:00 the regular meeting will start
At 16:00 the update will start, practicing the presentation and giving comments


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