Team:Aachen/Notebook/Wetlab/May

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Revision as of 19:45, 14 October 2014

May

1st

  • A gel with M - full -full - REACh1 SOE3.2 - REACH2 SOE3.2 - M was run
    → 120 V, 30 min
    → cut out the bands

5th

  • Chemical competent DH5α and BL21 cells were made

8th

  • The efficiency of our competent cells was tested
    → BL21: 6.6 x 104 clones/µg DNA
    → DH5α: 2.59 x 107 clones/µg DNA
  • SOE-PCR step 2 like on 30.04. with the template of SOE1 from 30.04. was re-done
  • The SOE2 product was run on a gel for checking (5 µL)
    → restriction, (dephosphorylation of vector)
    → purification on a gel with high pure kit

14th

  • LB agar plates with chloramphenicol and some with ampicillin were made
  • REACh2 was purified on 1.2 % agarose gel
  • A subsequent purification of the 778 bp fragment with High Pure PCR Product Purification Kit was done

19th

  • K131026 (AHL inducible GFP) was transformed into DH5α and NEB
  • K731520 (IPTG inducible GFPmut3b) was transformed into DH5α

20th

  • Master plates on chloramphenicol (cam) → at least 6 clones on each plate
  • 2x 5 mL LB + cam were prepared
  • Sterile 50 % glycerol was made

21th

  • Cryo stocks of clones 1 & 2 of each BioBrick/ host were prepared
  • 15 mL cultures in 250 mL flasks, inoculated with 1.5 mL preculture (3 cultures A B C from clones 1; 1 + 2; 2)
  • 250 µl Chloramphilicol from a 35 mg/mL stock was added
  • The cultures were grown until OD600= 0.6, then one was induced with IPTG
time info/ OD
11:10A: inoculated B: inoculated C: inoculated
11:38A: 0.482 B: 0.464 C: 0.466
12:28A: 0.586; induced with 0.1 mM IPTG B: 0.576 C: 0.568
14:11A: 0.93 B: 0.91 C: 0.942

→ A negative control without plasmid was left out

23rd

  • Some BioBricks were transformed
  • Colony PCR (s. picture below)
    → K131026: 1807 bp
    → K731520: 2123 bp
    → full length EYFP: 778 bp
Aachen 14-05-23 COLONY-PCR.jpg
Colony PCR
todo