IGEM:University of Groningen/2011/Notebook/Count coli/2011/08/08

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Count Coli <html><img src="/images/9/94/Report.png" border="0" /></html> Project description
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Mon 8 August 2011

Main Goals:

  • Constrution of autoinducing loops
  • Construction of internal control

Wet labwork

Cloning revP(BAD)+DT (insert) into pSB1K3-revDT+P(LasR)+LasR+TAG (vector)

  • PCR part --> revP(BAD)+DT from pSB1A3-revP(BAD)-DT using biobrick primer set
  • clone into pSB1K3-revDT+P(LasR)+LasR+TAG (12 different variants) on LB+kanamycine plate
  • Double digestion --> vector with SP, insert with XP
  • Ligate at 4 degrees ON

Cloning RBS-RFP (insert) into pSB1A3-DT (vector)

  • PCR part RBS-RFP from distributed DNA plasmid using 29F RBS-RFP and BB suffix primer set
  • clone to pSB1A3-DT on LB+ampicilin plate
  • Double digestion --> vector with EX, insert with ES
  • Ligate at 4 degrees ON


PCR Master Mix
37.71 μl MQ
5 μl 10X Taq buffer
3 μl MgCl2 (25mM)
1 μl dNTPs (10mM)
1 μl Forward (10μM)
1 μl Reverse (10μM)
0.25 μl Taq (5U/μl)
0.04 μl Pfu
1 μl Template DNA
50 μl Total reaction

PCR Results

  • revP(BAD)+DT --> proper size band!
  • RBS-RFP --> proper size band!

Double digestion mix ---incubate ON at 37 degrees
3 μl 10X FD buffer
1 μl enzyme #1
1 μl enzyme #2
1 μl alkaline phosphatase
vector (1μg)/insert (200ng)
add up to 30 μl with MQ

Notes

  • ON culture P(hybB)+RBS-GFP+DT --> NOT grow


Dry labwork

  • Kanamycine resistance cassete sequences analysis
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