Team:Heidelberg/Notebook/Sensing Group/Notebook/1stweek

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=== Preparations ===
=== Preparations ===
Preparations were started at the end of the week before
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)
+
* Transformation of pDK48 and pTrc99alpha plamids in ''E. coli'' strain DH5α (0.5 µl plasmid-DNA + 50 µl competent cells)
* Glycerol-stock of ''Vibrio harveyi'' BB120, BB886, mm30, BB178, BB125
* 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
+
* pick pTrc99α and pDK48 colonies and then incubated in 3 ml LB at 37 overnight
* ''V. harveyi'' cultured on LB plate
* ''V. harveyi'' cultured on LB plate
-
* MiniPrep of pTrc99a and pDK48 from DH5alpha
+
* MiniPrep of pTrc99α and pDK48 from DH5α
== Tuesday, 08/05/2008 ==
== Tuesday, 08/05/2008 ==
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[[Image:HD_080805-LuxP_PCR.png|thumb|150px|LuxP colony PCR]]
[[Image:HD_080805-LuxP_PCR.png|thumb|150px|LuxP colony PCR]]
<div style="float: right; clear:none;">[[Image:HD 080805-LuxQ S PCR.png|right|thumb|200px|LuxQ, LuxS PCR]]</div>
<div style="float: right; clear:none;">[[Image:HD 080805-LuxQ S PCR.png|right|thumb|200px|LuxQ, LuxS PCR]]</div>
 +
* 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)
* 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 H<sub>2</sub>O
* Gel Purification of LuxP eluted in 30 µl H<sub>2</sub>O
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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 (NEBuffer 3 + BSA)
+
* digestion of LuxS and pTrc99α 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
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== Friday, 08/08/2008 ==
== Friday, 08/08/2008 ==
* Miniprep of overnight-cultures of the picked colonies Q#6-15
* Miniprep of overnight-cultures of the picked colonies Q#6-15
-
* In silico cloning of LuxQ, S in pTrc99a and LuxP in pDK48
+
* In silico cloning of LuxQ, S in pTrc99α and LuxP in pDK48
* Test-Digestion of LuxQ with XbaI --> positive result for Q3, 7, 9, 11, 14
* Test-Digestion of LuxQ with XbaI --> positive result for Q3, 7, 9, 11, 14
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/2ndweek | Go to 2nd week]]
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/2ndweek | Go to 2nd week]]

Latest revision as of 14:01, 29 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 DH5α (0.5 µl plasmid-DNA + 50 µl competent cells)
  • Glycerol-stock of Vibrio harveyi BB120, BB886, mm30, BB178, BB125
  • pick pTrc99α and pDK48 colonies and then incubated in 3 ml LB at 37 overnight
  • V. harveyi cultured on LB plate
  • MiniPrep of pTrc99α and pDK48 from DH5α

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 pTrc99α 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 pTrc99α and LuxP in pDK48
  • Test-Digestion of LuxQ with XbaI --> positive result for Q3, 7, 9, 11, 14


Go to 2nd week

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