Joyce
Restriction analysis:
2μl FD buffer
0.5μl EcoRI
0.5μl PstI
7μl MQ water
10μl mix+ 10μl sample
Mix 2:
8μl FD buffer
2μl EcoRI
2μl PstI
28μl MQ water
10μl mix+ 10μl sample
Digestion vector:
4μl FD buffer
1μl EcoRI
1μl PstI
14μl MQ water
10μl mix+ 10μl sample
Digestion insert, PBAD, PcI, PlasB:
4μl FD buffer
1μl EcoRI
1μl PstI
14μl MQ water
10μl mix+ 10μl sample
Digest for 1h at 37 degrees
Analyse PhybB-RBS-GFP-DT on 1% TBE agarose gel
Ligate other digested DNA fragments with other promotors in vector with RBS-GFP-DT
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
5μl vector
6μl insert
6μl MQ water
Ligate for 2hours 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
Jakub
Week 26:
- PCR with different LasR variants
- Cloning LasR variants into plasmids with reverse double terminator and LasB promoter - first try.
Christoph
- Cloning the cI variants into pSB1A3+DT+rev.PlasB again...
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