IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-12

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Contents

Plasmid Construction

Transformation Result

  • R0010, pSB3K3 make it to grow. (Stored at 4 centigrade degree.)
  • However, pSB1A3 did not make it to grow (the second time). Possible Reason: Yu Tao made a big mistake -- He grew the transformant on a used plate.

Test the potency of competent cells made at 9th July

  • Transform 3uL proT into DH5alpha competent cells made at 9th July.
  • Competent cells made with the standard protocol: ~4000 (number of transformants).
  • Competent with an extra centrifugation during CaCl2 treatment: ~1300.

Fetch E0040

Another Tranformation

  • Transform 1uL E0040 and 1uL pSB1A3 into Mach1-T1 competent cells. (Thanks for the help from Switch group!)
  • Incubate in 37 centigrade degree.

Culture I14032, J23066, J61003, pSB1AK3, R0010 and pSB3K3 in Liquid LB (for miniprep tomorrow).

  • Antibiotics such as ampicillin and Kanamycin are added correspondingly to the LB media for each biobrick.

Streak the strain JM109 onto LB negative media

Plan for tommorow

  • Miniprep of the 6 successfully transformed biobricks
  • Making competent cells.

oriT Knock Out

  • By Xu Anting

Design Principle

  • Plasmid pKO3 is used as a tool for precisely oriT deletion. Please refer to our protocol page and Link A. et al., (1997) J Bacteriol, Vol. 179, No. 20, 6228–6237

PCR for oriT-Deleted fragments

  • Primers for cloning oriT-deleted fragments arrived.
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