Team:Heidelberg/Templates/MM week6

From 2013.igem.org

Contents

2013-06-03

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:
Cycles temperature [°C] Time [s]
1 98 30
30 98 10
66 30
72 90
1 72 450
1 4 inf
  • 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:
Cycles temperature [°C] Time [s]
1 98 30
5 98 10
39 45
72 90
5 98 10
45 45
72 90
20 98 10
58 45
72 90
1 4 inf
  • 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:
Cycles temperature [°C] Time [s]
1 98 30
35 98 10
40 60
72 120
1 72 600
1 4 inf
  • using hot start at 98°C
  • amplificate has 1.3kb -> too short, but specific (one distinct band)