Team:Heidelberg/Templates/MM week13p

From 2013.igem.org

Contents

2013-07-22

  • transform BBa_I746200 in pSB1AK3 (Spring 2012 Distribution Plate 2 Well 15E) in TOP10, plate on Amp
  • transform BBa_J04450 in pSB3C5 (Spring 2012 Distribution Plate 1 Well 3C) in TOP10, plate on Cm

2013-07-23

  • pick TOP10-BBa_I746200 colony, inoculate LB+Kan, grow at 37°C
  • pick TOP10-BBa_J04450 colony, inoculate LB+Cm, grow at 37°C
  • after 12 hours: add new antibiotics to medium, continue growing at 37°C

2013-07-24

2013-07-25

File:Methylmalonyl-PCR2013-07-25 1.png
Lane 1: NEB 2-log; lane 3: f1; lane 4: f2; lane 5: f3; lane 6: f4; lane 7: f5; lane 8: f6
  • run PCRs (using Q5, 20 µl total volume) using 4 ng of each template (pLF03 miniPrep from 2013-06-11, 21 ng/µl), sfp from Konrad's BAP1 plate:
Cycles temperature [°C] Time [s]
1 98 30 (f1,f2,f3,f5,f6) / 300 (f4)
35 98 5
66 (f1,f2) / 60 (f4) / 61 (f3,f5,f6) 10
72 120 (f1,f2,f3,f5,f6) / 30(f4)
1 72 600
1 12 inf
  • did not manage to get f3, f4, f6
  • repeat: add 1 µl DMSO to reactions:
File:Methylmalonyl-PCR2013-07-25 2.png
Lane 1: NEB 2-log; lane 2: f3; lane 3: f4 with lots of bacteria from new plate; lane 4: f4 with lots of bacteria from old plate; lane 5: f4 with little bacteria from new plate; lane 6: f6
Cycles temperature [°C] Time [s]
1 98 30
35 98 5
56 (f4) / 57 (f3,f6) 30
72 150 (f3,f6) / 45(f4)
1 72 600
1 10 inf
File:Methylmalonyl-PCR2013-07-25 3.png
Lane 1: NEB 2-log; lane 2: f3; lane 3: f6
  • use pLF03 miniPrep from 2013-06-11, 27 ng/µl, repeat for f3, f6 (with 1 µl DMSO):
Cycles temperature [°C] Time [s]
1 98 30
12 98 10
62 ↓0.5°C 10
72 150
23 98 10
58 10
72 150
1 72 600
1 10 inf

2013-07-26

File:Methylmalonyl-PCR2013-07-26.png
Lane 1: NEB 2-log; lane 2: f3 with annealing; lane 3: f6 with annealing; lane 4: f6 2-step; lane 5: f3 2-step
  • run PCR of f3, f6 with Phusion Flash (using pLF03 miniPrep from 2013-06-11, 21 ng/µl), without DMSO:
Cycles temperature [°C] Time [s]
1 98 10
35 98 1
65 5
72 75
1 72 600
1 10 inf
Cycles temperature [°C] Time [s]
1 98 10
5 98 1
65 5
72 75
30 98 1
72 75
1 72 600
1 10 inf
  • inoculate 10 ml LB+Amp with TOP10-pLF03, grow for 36h at 37°C, add Amp every 12 hours

2013-07-28

  • make miniPrep -> very low yield (ca. 1-2 ng/µl)
  • inoculate 6 ml LB+Amp with TOP10-pLF03, grow at 37°C