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oriT Knock Out

  • By Xu Anting and Liu Ting

What will you do after endless failure...?

  1. Cannot repeat the PCR result of oriT in F/R751/pSC101. In the four trials with various conditions, F bands appeared twice, R751 once, and pSC101 twice.
  • We tried all kinds of conditions and the damn PCR failed again and again! I guessed it was because of the unbalanced PCR primers. However scince I need a 30 bp overlap to link the two PCR fragments together (via overlapped PCR), I didn't have another choice in primers' sequences.
  1. I was tired of colony PCR and designed to do chromosome extraction and use them as template, to avoid as much protein (in bacteria) as possible.

Chromosome Extraction

  1. Picked a single colony from plates and shake overnight in 5 mL LB, 37 centigrade.
  2. As control, culture another sample in 30 centigrade (An unreasonable requirement from CGMCC).
  • Cells can grow up in LB medium with 37 centigrade, overnight. Still cannot make sure whether the conjugation plasmids are in the grown cells because of the PCR failure.

To-do List

Use chromosome extraction template, Taq and Pfu enzyme to try again. Plan to re-design primers in case it fails again.

Plasmid Construction

Eletrophoresis of Extracted Plasmid

  • By Ma and Yu Tao.
  • J23066, pSB3K3, pSB1AK3, R0010, J61003 are all tested. (Without Digestion.)
  • Each of R0010, J61003 and pSB1AK3 has a single band. (Weird) And pSB3K3 and J23066 have no bands.
  • Notes: This eletrophoresis is especially for me to better understand the essence of electrophoresis, to make better use of the DNA dye and to adjust the apparatus to a better condition. Besides, there is still something wrong with our UVP Bioimaging System. Therefore, the result of the electrophoresis cannot be shown.

Miniprep of E0040

  • BY Yu Tao.

Eletrophoresis of Extracted Plasmid

  • By Yu Tao.
  • R0040, J01060, E0040 adn B0015 are tested. (Without Digestion.)
  • All have a single band.
  • Note:

Instruction of the DNA Dye

For conveniences, the DNA dye can be diluted 100 times by TAE first and stored at 4 centigrade degree. The final cencentration in the Sample can be 1000 times diluted.

Preculture E0040 J23066 and pSB3K3 for Miniprep Tomorrow=

  • By Yu Tao.