IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-13

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Contents

First Test of oriT Knock Out Primers

  • By Xu Anting
  • Colony PCR. Temperature gradient is 45℃ to 65℃, with a volume of 10 uL
  1. Set gradient temparature of 51-65 centigrade and used Pfu enzyme. No fragment received T__T
  2. Repeat PCR using Taq enzyme.
    1. Result: F primers have bands at 51℃, but R751 and pSC101 show negative results in two separate tests.
  3. Task tomorrow: change PCR machine and other conditions and repeat colony PCR again.

Transformation Result

  • By Tao
  • E0040 made it to grow.
  • However, pSB1A3 still remains "silent". Possible reason: it is supposed to carry ccdB.
  • We used the transformants of proT as a positive control. It grows very well.

Miniprep

  • By Zheng Qinsi
  • J23066, pSB3K3 and I14032 multiplied less than the other 3.
  • Employed the "EasyPure Mini Plasmid Purification Kit" of Transgen to extract the plasmids.
  • Plasmids stored at 20 centigrade degree.
  • Because we lack the DNA dye necessary for agarose electrophoresis, the digestion test has not been carried out.

Making competent cells

  • By Tao and Zheng
  • Very tired job!!
  • Totally produced about 70 tubes of competent cells, 100uL per tube.
  • Stored at -80 centigrade degree.
  • Transformed 2uL proT(concentration unknown) into the fresh competent cells. 100uL and 200uL out of 400uL culture of transformed cells were plated,respectively. 200uL out of 400uL culture of untransformed competent cells as negative control.

Plans for Tomorrow (Lock and Key / Tandem OriT)

  • Digestion test of the extracted plasmids if DNA dye comes.
  • Fetch the biobricks R0040 and xxx(I forgot what it is) and transform them.
  • Digestion and ligation if DNA dye comes.
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