Haynes Lab:Notebook/Engineering PC-TFs/2014/03/20

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Summary

  • Ligation with CMV and MV9 using DH5α-T and BL21 cells


  Ligation Negative Control
Insert DNA (X ng) 4μL none
Vector DNA (50 ng) 1μL same
2x Roche Rapid Ligation buffer 6.0 μl same
New England Biolabs T4 ligase 1.0 μl same
dH2O 0μL 0μL + Insert μL
  12.0 μL total    same
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at room temperature for 10 minutes. Repeat once again for BL21. 6μL of buffer was used because the total volume with no water exceeded 10μL.


  1. Calculate how many ng of insert you need to get a 2:1 ratio of insert molecules to 50 ng vector molecules
    X ng CMV = (bp CMV = 588 / bp MV9 = 5291) x 2 x 50 ng MV9 = 11.1132ng insert (CMV)
  2. Calculate how many μL of insert and vector you will need for each ligation:
    X μL CMV = 11.1132ng CMV ÷ CMV concentration = 2.767ng/μL = 4.016μL
    X μL vector = 50 ng MV9 ÷ MV9 concentration = 49.159ng/μL = 1.0171μL
  3. Set up your ligation reaction(s) in sterile 0.5mL tubes as shown here:



Transform bacteria with the ligated plasmids 30 minutes

  1. Warm selection agar plates at 37°C.
  2. Incubate DH5α Turbo competent cells on ice just until thawed. Use 50 μL per ligation.
  3. Add 50 μL thawed cells to the ligation reaction. Immediately place on ice and incubate for 10 min. (Do not heat shock; No 30 min. recovery is required for Amp resistance)
  4. Label the pre-warmed plates with the antibiotic name, strain name, ligation (e.g., "BB part A insert + BB part B vector"), your initials, and the date.
  5. Pipette the total volume of cells + ligation onto the agar; spread using sterile glass beads.
  6. Incubate overnight at 37°C to get colonies

Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of vector self-ligation or selection failure. </div>

'Transforming bacteria with the ligated plasmids in BL21'

  1. Warm selection agar plates at 37°C.
  2. Incubate BL21 competent cells on ice just until thawed. Use 50 μL per ligation.
  3. Add 50 μL thawed cells to the ligation reaction. Immediately place on ice and incubate for 10 min.
  4. Heat shock at 45°C for 45 seconds.
  5. Immediately place on ice for 5 minutes.
  6. Add 750μL SOC medium to two new 1.5mL autoclaved tubes. Label.
  7. Transfer all cells and DNA to respectively labeled SOC medium tube. Place back on ice.
  8. Tape both tubes horizontally to the shaking part (great science nomenclature) of the incubator for 30 minute recovery.
  9. Pellet cells by centrifuging at max rpm for three minutes.
  10. Pipette out most of the SOC solution but leave a bit to re-suspend the cells.
  11. Pipette volume onto labeled and warmed agar plates. Spread using sterile glass beads.
  12. Incubate overnight at 37°C to get colonies


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