IGEM:Groningen/Notebook/iGEM 2011/2011/08/01

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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



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