IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-12
From OpenWetWare
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 [math]\displaystyle{ CaCl_2 }[/math] 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.
