Team:Paris/July 15

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(New page: {{Paris/Calendar_Links|July 14|July 16}} ==Preparation of Competent Cells== * Dilution 1/200e (1mL of the O/N culture on 200mL of LB without Antiobiotic) * Culture at 37°C until DO=0,46...)
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==Culture of Cells==
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* Culture of DH5ɑ in 10ml of LB without Antibiotics, O/N at 37°C

Revision as of 10:08, 16 July 2008

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Contents

Preparation of Competent Cells

  • Dilution 1/200e (1mL of the O/N culture on 200mL of LB without Antiobiotic)
  • Culture at 37°C until DO=0,460 (around 3h)
  • Sharing into 4 Falcon tubes of 50mL and cooling at 4°C during 40' in ice
  • Spin during 8', 2000rpm, 4°C
  • Resuspension of the cell's pellet in 20mL of cold CaCl2 (0,1M) during 30' in ice
  • Spin during 8', 2000rpm, 4°C
  • Resuspension of the cell's pellet in 1mL of cold CaCl2 (0,1M)


DNA Extraction of the Biobricks from the registery 2008

  • Warming of 10µl of TE at 50°C (in a 1.5ml tube)
  • Incubation of "the blotter" (recover by punch tool) during 5min at 50°C in the warm TE
  • Incubation 15min at RT (~30°C)
  • Recovering of 5µl plasmid


Transformation of the cells

  • Mix 150µl of competent cells + 5µl of DNA
  • Incubate 30min at 4°C on ice
  • Add 200µl of SOC, incubate 1h at 37°C
  • Spin 5min, at 2.000rpm, at RT
  • Remove 200µl of the supernatant, and resuspend the pellet in 150µl of the supernatant left
  • Spread all the suspension on plates with the adaptate antibiotics
  • Incubate O/N at 37°C


List of the transformations

  • Biobricks 2007
Miniprep Details Results
MP1 J23100 Transformation
MP2 J23107 Transformation
  • Biobricks 2008
Ref. Book Plasmid Antibio Plate 2007 Results
2E_SP 1000 B0034 A 3O P1 No transformation
10F_SP 1001 R0079 A 19N P1 No transformation
4F_SP 1013 C0079 K 7K P2 No transformation
6H_SP 1001 C0179 A 6H P1 No transformation
6D_SP 1007 S03879 A / No transformation
6B_SP 1001 S03154 A 24K P1 No transformation
10F_SP 1002 J23101 A 21G P3 No transformation
11F_SP 1002 J23109 A 23G P3 No transformation
4C_SP 1000 R0040 A 7O P1 No transformation
2G_SP 1000 C0040 A 5C P1 No transformation
1C_SP 1008 PSB1A3 A 23E P1 No transformation
7G_SP 1013 E1010 K 15M P2 No transformation
7E_SP 1001 E0040 A 5H P1 No transformation
2F_SP 1016 E0030 K 7G P1 No transformation


Culture of Cells

  • Culture of DH5ɑ in 10ml of LB without Antibiotics, O/N at 37°C
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