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  
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* digestion of LuxS and pTrc99a with BamHI/NcoI  
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* 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

Back to the overview

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

LuxP colony PCR
LuxQ, LuxS PCR
  • 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)
Colony PCR of LuxQ, LuxS and LuxP
  • 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
PCR to confirm insert from Minipreps. LuxQ no. 3 seems to be posivite. For LuxS and LuxP all samples are positive.
  • 10 new colonies of LuxQ-plate were picked and checked via colony-PCR
LuxQ colony PCR of 10 new colonies.
  • 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


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