Team:Heidelberg/Templates/MM week6

From 2013.igem.org

2013-06-03

• inoculate 50 ml LB with BAP1, grow at 37°C until OD=0.57
• prepare BAP1 glycerol stocks
• prepare competent BAP1
• recover pKD46, pCP20, pLF03
• transform TOP10 with pKD46, plate on Amp, grow at 30°C
• transform TOP10 with pLF03, plate on Amp, grow at 37°C
• transform TOP10 with pCP20, plate on Amp, grow at 30°C

2013-06-04

• inoculate 4 ml LB+Amp with TOP10-pLF03, grow at 37°C
• make miniPrep of pLF03 => 97 ng/µl in 27 µl
• transform BAP1 with pKD46, plate on Amp, grow at 30°C

2013-06-05

• prepare 50 ml LB + Amp + Arabinose(0.1%), inoculate with BAP1-pKD46
• prepare 4 ml LB + Amp, inoculate with TOP10-pKD46 (for miniPrep)
• no colonies with pCP20 => repeat transformation with three times the amount of plasmid (30 µl)
• BAP1-pKD46 reached OD=0.68 at 22:30 => put in fridge over night

2013-06-06

• inoculate 50 ml LB + Amp + Arabinose(0.1%) with 500 µl BAP1-pKD46 from ON culture (fridge)
• at OD=0.49: make glycerol stocks, prepare electrocompetent BAP1-pKD46
• make miniPrep of TOP10-pKD46 ON culture -> 31 ng/µl
• low yield of TOP10 miniPrep: make 2 miniPreps of BAP1-pKD46 ON culture -> 45.5 ng/µl and 46.0 ng/µl
• evening: no colonies with pCP20 -> write Lei Fang (Pfeifer lab) -> their stock is bad, we should get pCP20 somewhere else

2013-06-07

Gel electrophoresis of the PCR amplificate. Left: NEB 2 log DNA ladder; middle: PCR without annealing step; right: full PCR

• run PCR of 5 ng pLF03 (1 µl 1:20 dilution of miniPrep from 2013-06-04) of with primers ygfG21C1 and ygfG21C2 using Phusion polymerase:

• using hot start at 98°C
• two PCR samples were run, in one the annealing step at 66°C was left out
• => no amplificate

2013-06-08

Gel electrophoresis of the PCR amplificate. Left: NEB 2 log DNA ladder; middle: full PCR; right: PCR without 58°C annealing step

• Looking in more detail at the primers, the parts binding to the template have melting temperatures of 40 and 45°C (the rest is homologous to E. coli genome for recombination)
• run PCR of 5 ng pLF03 (1 µl 1:20 dilution of miniPrep from 2013-06-04) with primers ygfG21C1 and ygfG21C2 using Phusion polymerase:

• using hot start at 98°C
• two PCR samples were run, in one the annealing step at 58°C was left out
• => no amplificate

2013-06-09

Gel electrophoresis of the PCR amplificate. Left: NEB 2 log DNA ladder; right: PCR product

• run PCR of 5 ng pLF03 (1 µl 1:20 dilution of miniPrep from 2013-06-04) with primers ygfG21C1 and ygfG21C2 using Phusion polymerase:

• using hot start at 98°C
• amplificate has 1.3kb -> too short, but specific (one distinct band)