IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-16

=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.