IGEM:Groningen/Notebook/iGEM 2011/2011/08/22

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22-8-11



After rechecking: constructs with cI-LVA and LasR-LVA are not alright (in total 6 constructs...:( )
Doing it now over again with definetely the right primer set:

PCR on templates with cI-LVA and LasR-LVA:
Composition per reaction:
10× pfu buffer with MgSO4: 5μl
dNTPs 10mM: 1μl
Forward primer: 1μl
Reverse : 1μl
Template: 1μl
Pfu polymerase: 1μl
MQ water: 40μl

PCR conditions:
Pre heated lid: 111°C
Denaturation: 94°C for 3min
Cycle (33×)
Denaturation: 94°C for 30s
Annealing: 67°C for 30s
Extenstion: 72°C for 2min
Final extension: 72°C for 10min
Store infinite at 4°C
Analyse PCR samples on a 1% agarosegel

Meanwhile:
Purify PCR products and check with nanodrop the DNA concentration
After check, digestion:

------------
PCR did not go well. Do an overnight PCR again and everything from digestion to below will be done tomorrow

PCR on templates with cI-LVA and LasR-LVA:
Composition per reaction:
10× pfu buffer with MgSO4: 5μl
dNTPs 10mM: 1μl
Forward primer: 1μl
Reverse : 1μl
Template: 1μl
Pfu polymerase: 1μl
MQ water: 40μl

PCR conditions:
Pre heated lid: 111°C
Hotstart
Denaturation: 94°C for 5min
Cycle (33×)
Denaturation: 94°C for 30s
Annealing: 69°C for 30s
Extenstion: 72°C for 2min
Final extension: 72°C for 10min
Store infinite at 4°C
Analyse PCR samples on a 1% agarosegel




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

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

pSB1A3-DT vector
3μl vector
1μl XbaI
1μl PstI
3μl FD buffer
1μl FastAP
21μl MQ water

Incubate for 60 min at 37 degrees

DNA clean up:

Ligation:
cI-LVA-pSB1A3DT
8.5μl vector
7.1μl insert
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
1.4μl MQ water

LasR-LVA-pSB1A3DT
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

Incubate for 30-40 minutes 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