TOPO TA Cloning

Published on Author Giorgio Gilestro

Topo TA cloning is done following loose suggestions of the manufacturer. We currently adopt the kit K204040 from Invitrogen, which is not based on topoisomerase ligation but regular T4 ligation. Comes with “One Shot® aTOP10 Chemically Competent E. coli”.

Briefly.

  1. Quantify PCR product. The kit suggests to use 10ng of DNA. We usually skip this step.
  2. Mix water (5ul), 10X buffer (1ul), Vector (2ul),  T4 Ligase (1ul), PCR product (1ul). Make a master mix if you have more than one sample, then redistribute in PCR strip tubes as necessary and add the 1ul of PCR product at the end. Pipette up and down.
  3. Incubate overnight in the 18C fly room (protocols recommend 14C)
  4. The day after, warm up a water bath to 42C, warm up ampicillin plates. Add to each plate 40ul of a solution of X-GAL 40mg/ml in DMF. Warm up SOC medium (optional).
  5. Thaw bacteria in ICE (they are kept in my drawer in the -80C freezer). Each vial with purple cap should be used for one ligation reaction. Bacteria should never leave ice for longer than 4-5 seconds, unless they are being heat shocked.
  6. Add 2ul of ligation reaction to each LABELED tube and keep 30 minutes in ice.
  7. Heat shock for 30″ at 42C. Make sure, using a real thermometer, than the temperature of the water is really 42C.
  8. Put bacteria back to ice for a couple of minutes.
  9. (optional) Add 250ul of SOC to each tube and incubate at 37C for 1hour by shacking on the horizontal axis.
  10. Spread 20ul of SOC grown bacteria or otherwise spread the entire thing if you didn’t do SOC recovery.
  11. Put plates at 37C overnight. The day after pick white colonies only. Blue colonies are the reclosed vector.

 

PCR2.1 Multi Cloning Site