Team:Heidelberg/Templates/MM week22p

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Revision as of 17:13, 3 October 2013

Contents

2013-09-23

2013-09-27

File:Methylmalonyl-digest-PCR2013-09-27.png
Lane 1: NEB 2-log; lane 3: pIK8.6 digested with EcoRI+XbaI; lane 5: PCR of pIK8.6 with primers VF2+IK44
  • need to remove XbaI site for submission (for Boston) -> pIK12: amplify pccB-accA2 with primer introducing SpeI, ligate SpeI+Xba+ sites
  • run PCR of pIK8.6 with primers VF2+IK44 (3 ng DNA, 20 µl total volume, using Q5) -> f9:
Cycles temperature [°C] Time [s]
1 98 30
35 98 10
55 10
72 180
1 72 600
1 10 inf
  • digest 867 ng pIK8.6 (3 µl of miniPrep from 2013-09-15) with EcoRI+XbaI (20 µl total volume, using 0.5 µl enzyme), treat with antarctic phosphatase (37°C, 60 min)
  • gel-purify large pIK8.6 fragment, f9 (elute f9 in 17 µl)

2013-09-28

  • digest f9 with EcoRI+SpeI (0.5 µl enzyme, 20 µl total volume), purify with Nucleotide Removal Kit
  • ligate at RT for 1h:
what µl
pIK8.6 8
f9 9
T4 ligase 1 µl
T4 ligase buffer 2 µl
  • heat-inactivate at 75°C for 5 min
  • transform 10 µl into TOP10, plate 10 µl, rest on Cm, grow at 37°C

2013-09-28

  • lots of small colonies on both plates, several large colonies on both plates
  • pick 4 small, 1 large colonies from 10 µl plate, grow in 2xYT+Cm at 37°C
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