Team:Heidelberg/Templates/MM week15

From 2013.igem.org

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m (Created page with "== 2013-08-05 == * run colony-PCR against sfp on BAP1-pKD46 with primers RB43+RB44 (iTaq, 20 µl total volume): [[File:BAP1-colony-PCR2013-08-05.png|100px|thumb|right|Colony-PCR ...")
 
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== 2013-08-05 ==
== 2013-08-05 ==
* run colony-PCR against sfp on BAP1-pKD46 with primers RB43+RB44 (iTaq, 20 µl total volume):
* run colony-PCR against sfp on BAP1-pKD46 with primers RB43+RB44 (iTaq, 20 µl total volume):
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[[File:BAP1-colony-PCR2013-08-05.png|100px|thumb|right|Colony-PCR of BAP1-pKD46 with primers RB43+RB44. Lane 1: sfp, lane 2: NEB 2-log]]
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[[File:Heidelberg_BAP1-colony-PCR2013-08-05.png|100px|thumb| Colony-PCR of BAP1-pKD46 with primers RB43+RB44. Lane 1: sfp, lane 2: NEB 2-log]]
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== 2013-08-06 ==
== 2013-08-06 ==
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[[File:Methylmalonyl-PCR2013-08-06_2.png|100px|thumb|left|Lane 1: NEB 2-log; lane 3: colony-PCR with primers RB43+RB44]]
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[[File:Heidelberg_Methylmalonyl-PCR2013-08-06_2.png|100px|thumb| Lane 1: NEB 2-log; lane 3: colony-PCR with primers RB43+RB44]]
* inoculate 50 ml 2xYT+Amp+Ara(0.5%) with BAP1-pKD46, grow at 30°C until OD=0.71
* inoculate 50 ml 2xYT+Amp+Ara(0.5%) with BAP1-pKD46, grow at 30°C until OD=0.71
* run colony-PCR with primers RB43+RB44 from 50 ml culture:
* run colony-PCR with primers RB43+RB44 from 50 ml culture:

Latest revision as of 02:36, 5 October 2013

2013-08-05

  • run colony-PCR against sfp on BAP1-pKD46 with primers RB43+RB44 (iTaq, 20 µl total volume):
Colony-PCR of BAP1-pKD46 with primers RB43+RB44. Lane 1: sfp, lane 2: NEB 2-log
Cycles temperature [°C] Time [s]
1 95 300
35 95 60
55 30
72 180
1 72 600
1 10 inf
  • inoculate 4 ml 2xYT+Amp with BAP1-pKD46, grow at 30°C

2013-08-06

Lane 1: NEB 2-log; lane 3: colony-PCR with primers RB43+RB44
  • inoculate 50 ml 2xYT+Amp+Ara(0.5%) with BAP1-pKD46, grow at 30°C until OD=0.71
  • run colony-PCR with primers RB43+RB44 from 50 ml culture:
Cycles temperature [°C] Time [s]
1 95 300
35 95 60
55 30
72 180
1 72 600
1 10 inf

2013-08-08

  • electroporate BAP1-pKD46 with 500 ng DNA (5 µl of PCR amplificate from 2013-07-08)
  • grow in SOC + IPTG(1mM) + Arabinose (0.5%) at 37°C, 400 rpm for 3h
  • spin cells down (3 min at 8000 rcf)
  • plate 10 µl, rest on Cm, grow at 37°C