IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-5

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Contents

Tandem OriT by Qu Mingzhi and Yang Yifan

Bacteria Mating

  • By Yang Yifan
  • Donnor: E.coli strain C600 with plasmid R751 and pSC101, use cells with only R751 as negative control;

Recipient: E.coli strain DH5α with plasmid pSB1A2 confering ampicilin resistance.

  • Recipient are grown to stationary phase, Donnor are grown to log phase and stationary phase in two independent experiments.
  • OD600: recipient - 1.118AU; donnor(stationary) - 1.308AU; donnor(log-phase) - 0.524AU; donnor(control) - AU.
  • 500ul culture centrifuge and resuspension with LB for two times to wash the antibiotics.
  • Mix, and culture under 37°C for 90 min. Culture two more stationary-stationary mixture for 60min and 120min as a time control.
  • Plating with 200ul.

Amplification Culture of F-OriT_pSB1A2 <- J23066

  • by Qu Mingzhi
  • Culture at Amp+ plate for 12 hours.
  • select Positive F-OriT_J23066_pSB1A2 Colonies from Plate, Culture in liquid LB,waiting for mini-prep.
  • F-OriT_pSB1A2 self-ligation nagetive-control received 10 clones.

Lock & Key By Yu Tao

oriT Knock Out

  • By Xu Anting

oriT-deleted fragment

  • 0.8% agarose gel purification: R751-No.3 has a digested fragment of 750 bp. Repeat the same amplification-digestion experiment using R751-No.4.
  • pKO3 vector purification fails... Re-amplify pKO3 in 30℃ for 24 hours.
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