Team:Aachen/Notebook/Wetlab/May

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May

1st

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

5th

  • made chemical competent DH5α and BL21 cells

8th

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

14th

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

19th

  • transformation of K131026 in DH5α and NEB
  • transformation of K731520 in DH5α

20th

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

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

  • transformation of some BioBricks
  • 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