IGEM:IMPERIAL/2006/LabCalendar/2006-8-1

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Contents

Mini-prep parts

  • 7A->3O not successful first mini-prep, Dr. Mann suggested redoing ligation. However, upon second attempt to mini-prep, one colony appears to have successfully taken up the ligated vector (Gel E, Lane 2)
  • 12D->2H unsucessful ligation according to miniprep results (Gel B and Gel C lanes 2 and 3). Need to religate tomorrow.
  • 12D->24A not successful first mini-prep, redoing miniprep this afternoon. Successful mini-prep results can be seen in Gel C (Lanes 4 and 5) and Gel D (Lanes 2 and 3).

Re-ligation of 7A->3O

  • Digests
    • 7A with X & P, using yellow buffer
    • 3O with S & P, using orange buffer
  • Run on large gel
    • Successful...was able to cut out insert and vector
    • 7A extracted 756 bp (insert)
    • 3O extracted 2091 bp (vector)
  • Preform ligation after gene cleaning
    • 16 uL insert
    • 1 uL vector
    • 2 uL ligase buffer
    • 1 uL ligase

M9 broth

  • We want a total of 200 mL
    • 100 mL 2x M9 salts
    • 6 mL 10 mg/mL thiamine
    • 2 mL 40% glycerol
    • 4 mL 10% casamino acids
    • 4 mL 0.1 M MgSO4
    • 40 uL 0.5M CaCl2
    • Make up to 200 mL with sterile water
  • See Endy Lab Protocol on OWW

Electroporations Tomorrow

  • 7A->3O ligation
  • Part I13521 well 18I, plate 1

Maxi-prep tomorrow

  • 12D->2H
  • J04500, 16P
  • C0060, 5M
  • B0015, 1I
  • Recultured bacteria in 100 mL LB with Amp with 100 uL bacteria culture
  • Set for at least 14 hours in 37C shaker
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