Team:Heidelberg/Notebook/Sensing Group/Notebook/5thweek

From 2008.igem.org

(Difference between revisions)
(Monday, 09/01/2008)
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== Monday, 09/01/2008 ==
== Monday, 09/01/2008 ==
 +
* 1.selection for Fusion-1 and Fusion-2 cloning
 +
* the DNA of the colonies was extracted with MINIprep, and then screen with test digestion: PstI (Buffer3+BSA at 37)
 +
** for Fusion-1: if our insert is there: one band of 6101 bp if no inser: two bands: 4388 + 2213 bp
 +
** for Fusion-2: if our insert is there: one band of 6122 bp if no inser: two bands: 4388 + 2213 bp
 +
 +
PCR of LuxQ and two fusion protein constructs
 +
* Lyse ''V. harveyi'' for 5 min @ 99 °C
 +
* PCR with Phusion (template: supernatend of boiled ''V. harveyi'' (a), colony (b))
 +
** 5 min @ 98 °C || 20s @ 98 °C | 40s @ 58 °C | 1min @ 72 °C || 10 min @ 72 °C | 4 °C hold ( 35 cycles )
 +
** 35(34) µl H<sub>2</sub>O, 10 µl 5x HF, 0.5 µl each primer, 1µl Template, 2µl dNTPs, 1µl Phusion, 0(1) µl DMSO
 +
* PCR with Pfu
 +
** 3 min @ 94 °C || 45s @ 94 °C | 45s @ 58 °C | 2min @ 72 °C || 10 min @ 72 °C | 4 °C hold ( 35 cycles )
 +
** 40(39) µl H<sub>2</sub>O, 5 µl 10x Buffer, 0.5 µl each primer, 1µl Template, 2µl dNTPs, 1µl Pfu, 0(1) µl DMSO
 +
 +
'''NO PCR Product'''
== Tuesday, 09/02/2008 ==
== Tuesday, 09/02/2008 ==

Revision as of 15:46, 26 October 2008

Back to the overview

Contents

Monday, 09/01/2008

  • 1.selection for Fusion-1 and Fusion-2 cloning
  • the DNA of the colonies was extracted with MINIprep, and then screen with test digestion: PstI (Buffer3+BSA at 37)
    • for Fusion-1: if our insert is there: one band of 6101 bp if no inser: two bands: 4388 + 2213 bp
    • for Fusion-2: if our insert is there: one band of 6122 bp if no inser: two bands: 4388 + 2213 bp

PCR of LuxQ and two fusion protein constructs

  • Lyse V. harveyi for 5 min @ 99 °C
  • PCR with Phusion (template: supernatend of boiled V. harveyi (a), colony (b))
    • 5 min @ 98 °C || 20s @ 98 °C | 40s @ 58 °C | 1min @ 72 °C || 10 min @ 72 °C | 4 °C hold ( 35 cycles )
    • 35(34) µl H2O, 10 µl 5x HF, 0.5 µl each primer, 1µl Template, 2µl dNTPs, 1µl Phusion, 0(1) µl DMSO
  • PCR with Pfu
    • 3 min @ 94 °C || 45s @ 94 °C | 45s @ 58 °C | 2min @ 72 °C || 10 min @ 72 °C | 4 °C hold ( 35 cycles )
    • 40(39) µl H2O, 5 µl 10x Buffer, 0.5 µl each primer, 1µl Template, 2µl dNTPs, 1µl Pfu, 0(1) µl DMSO

NO PCR Product

Tuesday, 09/02/2008

Wednesday, 09/03/2008

Thursday, 09/04/2008

Friday, 09/05/2008

Go to 6th week