IGEM:Groningen/Notebook/iGEM 2011/2011/07/25

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25-7-11



Plates of transformants with PBAD-cI-LVA-DT and PBAD-RBS-GFP-DT vector look good, since the self ligation plates don't have much colonies!
10μl plate is empty on self ligation plate control! 90μl plate has a few colonies...
Colony PCR as usual-> PBAD-cI-LVA-DT looks OK-> grow colony 8 and 9 overnight.

Digestion PBAD, PhybB and vectors: cI-LVA and RBS-GFP

PBAD:
15μl PBAD
1μl SpeI
2μl Tango buffer
2μ MQ water

PhybB
3μl PhybB
1μl SpeI
2μl Tango buffer
14μl MQ water

After 2h: put 2.5μl Tango buffer+ 1μl EcoRI in these samples and digest for another 2h.
DNA clean up with kit of Roche.

Digestion vector (with LasR-LVA-DT):

3μl vector
1μl EcoRI
1μl XbaI
3μl Fast digest buffer
1μl Fast AP
21μl MQ water

Ligation:
PBAD-LasR-LVA-DT
6μl PBAD
8.5μl cI-LVA-DT vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
2.5μl MQ water

PhybB-LasR-LVA-DT
6μl PhybB
8.5μl cI-LVA-DT vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
2.5μl MQ water


Ligate for 30 to 45 minutes

Transformation as usual

Plasmid prep of LasR-LVA-DT colonies 6 and 7. DNA Concentration according to the N-D1000 Nanodrop is 46.7 and 51 ng/μl
respectively.
Colony 6 plasmid prep sample was used for vector sample today
Colonies 6 and 7 were send for sequencing and a glycerol stock has been made.

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