18 July 2013

From 2013.igem.org

TOPO TA cloning was performed for two PCR products, aptamers from SELEX cycle 11: one is with A-overhangs and another is without overhangs.
    Ligation reaction (7ul for 1 tube):
  • Fresh PCR product 3ul
  • Salt solution 1ul
  • Water 2ul
  • TOPO Vector 1ul
  1. Put 2 tubes of JM109 competent cells on ice (100ul each)
  2. Tap tubes to resuspend cells
  3. Add 7ul of ligation reaction to each cell tube, tap
  4. Incubate on ice for 30 min
  5. Heat-shock at 37degC for 45 seconds
  6. Immediately put on ice for 2 min
  7. Add 450ul of SOC medium and incubate at 37degC at 230rpm for 1 hour
  1. Prepare 8 flasks for incubation of transformed cells in LB broth (4 for each PCR product)
  2. Label: Ampicillin, Kanamycin, Control and Blank
  3. Add 5ml of LB broth to each flask
  4. Add to flasks, labeled for Ampicillin, 2.5ul of 50ug/ml Ampicillin
  5. Add to flasks, labeled for Kanamycin, 0.25ul of 1g/ml Kanamycin
  6. Add 40ul of 40mg/ml X-gal to each flask
  7. Add 40ul of 100mM IPTG
  8. Add 50ul of transformed cells to each except blanks
In addition, we plated cells that contain product with A-overhangs on 2 plates: with ampicillin and with kanamycin. We left everything for incubation overnight.