EXPERIMENTS

Competent Cell Preparation:


  • Grow E. coli DH5ɑ/BL21 culture in 5 mL of LB broth from a single colony from a plate for 12 hours at 37℃ and 120 rpm.
  • Inoculate 1% inoculum from the overnight culture into 20 mL of LB broth and grow till the OD600 is 0.4-0.5.
  • Harvest the cells by centrifuging the culture at 5,000 rpm for 10 mins at 4℃.
  • Wash the cell pellet with 5 mL of ice cold freshly prepared 75 mM CaCl2 solution.
  • Harvest the cells by centrifuging the culture at 5,000 rpm for 10 mins at 4℃.
  • Resuspend the pellet in 5 mL of ice cold CaCl2 solution and keep on ice for 30 mins.
  • Harvest the cells by centrifuging the culture at 5,000 rpm for 10 mins at 4℃.
  • Resuspend the pellet in 500 𝜇L of ice cold CaCl2 solution. Store on ice.
  • Cells will become competent after 16-19 hrs.

Restriction Digestion:


  • Combine the following in a PCR or Eppendorf tube:
    • DNA (ideally around 0.4 - 0.6 𝜇g)
    • 3 𝜇L Enzyme-specific 10x buffer
    • 1 𝜇L Restriction Enzyme
    • H2O to make up volume till 30 𝜇L
  • Incubate at 37℃ overnight

Gel Extraction:



  • Done using Qiagen’s QIAquick® Gel Extraction Kit. Alternate protocols may be there based on the kit used

Ligation:


  • Combine the following in a PCR or Eppendorf tube:
    • Vector DNA
    • Insert DNA
    • 3 𝜇L 10x Ligase Buffer
    • 0.5-1 𝜇L T4 DNA Ligase (depending on DNA concentration)
    • H2O to make up volume till 30 𝜇L
  • Incubate at room temperature at 16 - 18°C overnight

Transformation:


  • Add 3-5 𝜇L of the insert DNA to 50 𝜇L of competent cells. Keep on ice for 30 mins.
  • Give it a heat shock for 30 secs at 42℃.
  • Keep it on ice for 15 mins.
  • Add 1 mL of LB broth and incubate at 37℃ for 1 hr.
  • Harvest the cells at 2,500 rpm for 1-2 mins and resuspend the pellet 1/5th of the supernatant.
  • Spread on a LA antibiotic plate and incubate at 37℃.
  • Observe colonies after 12 hrs.

Plasmid Isolation:


  • Centrifuge 2 mL of overnight culture at 2000g for 10 mins at 4℃.
  • Discard the supernatant and resuspend the pellet in 100 𝜇L of ice cold Alkali Lysis Solution-I. Mix by vigorous vortexing.
  • Add 200 𝜇L of freshly prepared ice cold Alkali Lysis Solution-II. Mix by inverting the tube 5-6 times.
  • Immediately add 150 𝜇L of Alkali Lysis Solution-III. Disperse by rapidly inverting the tube several times. Incubate on ice for 5 mins.
  • Centrifuge at 1200 rpm for 6 mins at 4℃.
  • Transfer ~400 𝜇L of the supernatant to a fresh 1.5 ml eppendorf tube.
  • Add 1.5 𝜇L of RNase A. Incubate at 37℃ for 1 hr.
  • Add an equal volume of Phenol:Chloroform:Isoamyl alcohol (25:24:1). Mix by vigorous vortexing.
  • Centrifuge at 12,000 rpm for 3 mins at 4℃.
  • Transfer the aqueous layer to a fresh 1.5 mL eppendorf tube and add an equal volume of isopropanol at room temperature. Mix and allow to stand.
  • Centrifuge at 12,000 rpm for 6 mins at room temperature.
  • Discard the supernatant and add 250 𝜇L of 70% ethanol.
  • Centrifuge at 7,500 rpm for 5 mins at room temperature.
  • Air dry, then dissolve in 30 𝜇L nuclease-free water.