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

→ 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

Colony PCR todo