Team:Groningen/project notebook/23 August 2011

Joyce

Change of cloning strategy:
Instead of cloning 3 times, I can do a procedure with only 2 cloning steps since I already have the constructs PhybB and PBAD
in a chloramphenicol vector. So what I am going to do is:
Clone the RBS-cI-LVA and RBS-LasR-LVA in the vector with already PhybB and PBAD sequences, but than you have to clone
downstream those promotors the protein sequences. That is why you digest the vector with SpeI and PstI and the PCR products
with XbaI and PstI. If the transformation plates look well and the colony PCR can be done and show that there are some clones
having the right fragmentsize, thursday the plasmids can be isolated and the second cloning procedure can be done: cloning
downstream the double terminator sequence.
So if it all goes well, the problem will be fixed on friday

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

LasR-LVA
14μl insert
1μl XbaI
1μl PstI
2μl FD buffer
2μl MQ water

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

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

Incubate for 60 min at 37 degrees

DNA clean up:

Ligation:
PBAD-RBS-cI-LVA-pSB1C3
8.5μl vector
5.6μl insert
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
2.9μl MQ water

PBAD-RBS-LasR-LVA-pSB1C3
8.5μl vector
6.7μl insert
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
1.8μl MQ water

PhybB-RBS-cI-LVA-pSB1C3
8.5μl vector
7μl insert
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
1.5 μl MQ water

PhybB-RBS-LasR-LVA-pSB1C3
8.5μl vector
8.5μl insert
2μl T4 DNA ligase buffer
1μl T4 DNA ligase


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

PCR for the double terminator part:
Composition per reaction:
10× pfu buffer with MgSO4: 5μl
dNTPs 10mM: 1μl
Forward primer: 1μl
Reverse : 1μl
Template(pSB1A3DT): 1μl
Pfu polymerase: 1μl
MQ water: 40μl

PCR conditions:
Pre heated lid: 111°C
Denaturation: 94°C for 10min
Cycle (33×)
Denaturation: 94°C for 30s
Annealing: 60°C for 30s
Extenstion: 72°C for 1.5min
Final extension: 72°C for 15min
Store infinite at 4°C
Analyse PCR samples on a 1% agarosegel