IGEM:University of Groningen/2011/Notebook/Count coli/2011/08/09

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Count coli project description
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Tue 9 August 2011

To do List

  • Ligation and transformation --> 12 variants autoinducing loop + RFP internal control

Ligation mix---1 h incubation at RT
2 μl 10X FD buffer
1 μl T4 DNA ligase
vector (20ng)
insert

Implementation

1.revP(BAD)+DT into pSB1K3 revDT+P(LasR)+LasR
[insert] = 700 ng/50 μl = 14 ng/μl
For 1:6 vector to insert ration, insert mass = 62 ng ~ 4.4 μl


12 ligation reaction, each containing:
4.1 μl MQ
2 μl 10x buffer
1 μl T4 DNA ligase
8.5 μl vector
4.4 μl insert

2.RBS-RFP into pSB1A3-DT
[insert] = 222 ng/50 μl = 14 ng/μl
For 1:6 vector to insert ration, insert mass = 40 ng ~ 9 μl


Ligation mix
2 μl 10x buffer
1 μl T4 DNA ligase
8.5 μl vector
9 μl insert

Transformation Protocol

  • Add 10 μl ligation mixture to 40μl E.coli DH5alpha competent cells
  • Incubate on ice for 30 min
  • Heat shock at 42 degrees for 90 sec
  • Place tubes back on ice immediately for 10 minutes.
  • Add 1 ml LB without antibiotic
  • Incubate the culture at 37 degrees for 1 hour
  • Plate out the cell on agar plates with the appropiate antibiotic
  • Incubate the plates ON at 37 degrees.


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