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Team:Groningen/project notebook/25 July 2011
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JoyceM1013 (Talk | contribs) (Created page with "{{HeaderGroningen2011}} {{notebookgroningen}} '''Joyce''' <br> <br> Plates of transformants with PBAD-cI-LVA-DT and PBAD-RBS-GFP-DT vector look good, since the self ligation pl...") |
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<br> Colony 6 plasmid prep sample was used for vector sample today | <br> Colony 6 plasmid prep sample was used for vector sample today | ||
<br> Colonies 6 and 7 were send for sequencing and a glycerol stock has been made. | <br> Colonies 6 and 7 were send for sequencing and a glycerol stock has been made. | ||
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| + | '''Jakub''' | ||
| + | <br> | ||
| + | Week 30: | ||
| + | *Cloning LasR variants into plasmids with reverse double terminator and LasB promoter - third try. | ||
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{{FooterGroningen2011}} | {{FooterGroningen2011}} | ||
Revision as of 13:35, 21 September 2011
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Joyce
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.
Jakub
Week 30:
- Cloning LasR variants into plasmids with reverse double terminator and LasB promoter - third try.
