April

1st

Organization

Getting all kind of lab equipment

• key cards and keys for the lab
• cooled centifuge
• space reservation in -80°C freezer
• bottles for 70% ethanol
• ... (problems of a first year team ^^)

12th

• chemically competent E. coli NEB Top10, DH5α and BL21 were made

15th

• 800 ml LB for plates with 1.5% agar and kanamycin (50 µg/L) were prepared
• test of transformation efficiency of our competent cells
  • Colonies on the agar plates were counted after transformation with 1 µl DNA (147 ng/µl)
  • Cells had been incubated in SOC or LB medium

• BL21 cells were centrifuged and plated; only 128 colonies grew in total indicating a very bad efficiency (~482.11).

Following formula was used to calculate the efficiency: Efficiency = (CFU /µg DNA) / Dilution

• for DH5α: medial efficiency in SOC: 1864.36, and in LB: 440.68
• for NEB: medial efficiency in SOC: 6779.66, and in LB: 4698.30

  Higher efficiency when using SOC!

22th

Pcq lab PCR advanced primus 25, 96

1. RFC25_F RFC25_R
2. RFC25_F REACh1_R
3. REACh1_F RFC25_R
4. RFC25_F REACh2_R
5. REACh2_F RFC25_R

23th

• 5 µl of the PCR products were run on a 1,5% agarose gel
• DNA bands were purified from gel with the High Pure Product Purification Kit. Full length products were contaminated with REACh1

• SOE
• ca. 30 ng/µL

Anneling: 52°C

Elongation: 20 sec

• PCR with Phusion polymerase

• Digestion for restriction with NEB enzymes

• ligation with NEB Kit

28th

• The DNA concentration of the SOE2 products were measured after purification
• 1: 57 ng/µL
• 2: 69.5 ng/µL

29th

• PCR SOE1 and SOE2

30th

• A PAGE of the SOE2 products was run on agarose gel for checking

  → no positive results; only full length products were amplified

• SOE PCRs were run again

  → more template

  → hot start

  → faster

  → elongation: 15 min

  → 20 cycles

• SOE1

Template A+B → 20 µL → 13.8 + 6.2 5.3 + 14.7

• SOE2