Joyce
PBAD sample seems to be wrong. Received new one from iGEM Groningen 2009 team.
PCR PBADaraC:
10× Pfu buffer MgSO4: 5μl
dNTPs 10mM: 1μl
Forward primer 36F: 1μl
Reverse primer 37R: 1μl
(primers contain prefix and suffix)
template: 1μl
pfu polymerase: 1μl
MQ: 40μl
Checking PCR products on 1% agarosegel
PCR clean up with kit and measure DNA concentration with ND1000 nanodrop.
Digestion:
cI-LVA-DT
vector: 2μl
EcoRI: 1μl
XbaI: 1μl
FastAP: 1μl
Fast digest buffer: 3μl
MQ: 22μl
LasR-LVA-DT
vector: 3μl
EcoRI: 1μl
XbaI: 1μl
FastAP: 1μl
Fast digest buffer: 3μl
MQ: 21μl
RBS-GFP-DT
vector: 3μl
EcoRI: 1μl
XbaI: 1μl
FastAP: 1μl
Fast digest buffer: 3μl
MQ: 21μl
PBADaraC
insert: 4μl
EcoRI: 1μl
SpeI: 1μl
Fast digest buffer: 2μl
MQ: 12μl
Digest for 1h at 37 degrees
DNA clean up
Ligation:
PBADaraC-cI-LVA-DT
Vector: 8.5μl
insert: 6.1μl
T4 DNA ligase: 1μl
T4 DNA ligase buffer: 2μl
MQ: 2.4μl
PBADaraC-LasR-LVA-DT
Vector: 8.5μl
insert: 6.1μl
T4 DNA ligase: 1μl
T4 DNA ligase buffer: 2μl
MQ: 2.4μl
PBADaraC-RBS-GFP-DT
Vector: 8.5μl
insert: 6.1μl
T4 DNA ligase: 1μl
T4 DNA ligase buffer: 2μl
MQ: 2.4μl
Selfligation tests:
Vector: 8.5μl
T4 DNA ligase: 1μl
T4 DNA ligase buffer: 2μl
MQ: 8.5μl
Ligate for 30 min at room temperature
Transformation as usual
Jakub
Week 31:
- Cloning LasR variants into plasmids with reverse double terminator and LasB promoter - third try and success.
Christoph
- Changed cloning strategy and got good constructs finally.
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