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Team:Heidelberg/Notebook/Sensing Group/Notebook/1stweek
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=== Cloning of LuxS === | === Cloning of LuxS === | ||
* PCR of ''V. harveyi'' genome with primers LuxSa and LuxSb and PCR purification 5min @ 95 °C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles) with Tag polymerase Mastermix | * PCR of ''V. harveyi'' genome with primers LuxSa and LuxSb and PCR purification 5min @ 95 °C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles) with Tag polymerase Mastermix | ||
| - | * digestion of LuxS and pTrc99a with BamHI/NcoI | + | * digestion of LuxS and pTrc99a with BamHI/NcoI (NEBuffer 3 + BSA) |
* Ligation with vector:insert ratio 2µl:6µl and 5µl:5µl | * Ligation with vector:insert ratio 2µl:6µl and 5µl:5µl | ||
Revision as of 23:32, 26 October 2008
Contents |
Monday, 08/04/2008
Preparations
Preparations were started at the end of the week before
- Transformation of pDK48 and pTrc99alpha plamids in E. coli strain DH5alpha (0.5 µl plasmid-DNA + 50 µl competent cells)
- Glycerol-stock of Vibrio harveyi BB120, BB886, mm30, BB178, BB125
- pick pTrc99a and pDK48 colonies and then incubated in 3 ml LB at 37 overnight
- V. harveyi cultured on LB plate
- MiniPrep of pTrc99a and pDK48 from DH5alpha
Tuesday, 08/05/2008
Cloning of LuxP
- PCR of LuxP from V. harveyi colony with the primers LuxPc and LuxPd with Taq polymerase Mastermix. 5min @ 95°C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles)
- Gel Purification of LuxP eluted in 30 µl H2O
- Digestion of LuxP with SalI and NotI of pDK48 and LuxP-Gel-Purification product: 2 µl NEB-Buffer 3 + 0.5 µl SalI & NotI + 10 µl DNA + 6 µl H2O
- Ligation with vector:insert ratio 2µl:6µl and 5µl:5µl
Cloning of LuxS
- PCR of V. harveyi genome with primers LuxSa and LuxSb and PCR purification 5min @ 95 °C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles) with Tag polymerase Mastermix
- digestion of LuxS and pTrc99a with BamHI/NcoI (NEBuffer 3 + BSA)
- Ligation with vector:insert ratio 2µl:6µl and 5µl:5µl
Wednesday, 08/06/2008
- 5 colonies of LuxQ,S,P transformation (1:1 ligation) picked and analyes with colony-PCR
- Colony-PCR to check for insert. 5min @ 95°C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles)
- preparation of O/N cultures for Miniprep
Thursday, 08/07/2008
- Miniprep of samples Q#1-5, S#1-5, P#1-5
- PCR from Minipreps to confirm inserts. Same programme as yesterday
- 10 new colonies of LuxQ-plate were picked and checked via colony-PCR
- Sequencing of LuxS,P,Q positive clones at GATC
Sequencing Results LuxS: correct sequence LuxP: all clones contain mutations, cloning has to be repeated LuxQ: no insert in the picked colonies
Friday, 08/08/2008
- Miniprep of overnight-cultures of the picked colonies Q#6-15
- In silico cloning of LuxQ, S in pTrc99a and LuxP in pDK48
- Test-Digestion of LuxQ with XbaI --> positive result for Q3, 7, 9, 11, 14
