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

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.

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.