Team:REC-CHENNAI/Protocols

Protocols

Home > Protocols

Competent Cells


Competent cells are typically E.coli that are capable of incorporating foreign genetic material into their nucleus. This is done by the Calcium Chloride- Heat shock technique. The principle behind the incorporation of this genetic material is the generation of a calcium rich environment which counteracts repulsion between plasmid DNA and bacterial cell membrane. Sudden exposure to heat forms pores on the bacterial cell membrane, allowing the entry of foreign genetic material.


Preparation of Competent Cells


  1. Add 1ml of overnight culture to 100ml of freshly prepared LB (Luria Bertani) broth.
  2. Incubate it in a shaker at 200 rpm at a temperature of 37°C until OD at 610nm which is between 0.4-0.6.
  3. Incubate at 4°C for 30 minutes. This process arrests the metabolism of the organism in the culture.
  4. Aliquot the sample into 6 autoclaved and chilled 15ml falcon tubes.
  5. Prechill the centrifuge and place the falcon tubes in it for centrifugation at 6500 rpm for 10 minutes at 4°C.
  6. Discard the supernatant carefully and resuspend the pellet in 4ml of freshly prepared and chilled CCMB buffer.
  7. Centrifuge at 6500 rpm and 4°C for 10 minutes.
  8. Discard the supernatant and resuspend the pellet in 2ml of chilled CCMB buffer.
  9. Centrifuge at 6500 rpm and 4°C for 10 minutes.
  10. Discard the supernatant and resuspend the pellet in 1ml of chilled CCMB buffer. Perform this step using autoclaved microfuge tubes.
  11. Centrifuge the resuspended pellet at 6500 rpm and 4°C for 10 minutes.
  12. Discard the supernatant and resuspend the pellet in 100µl of chilled CCMB buffer.

Precautions


  1. Autoclave glassware, pipette tips, falcon and microfuge tubes before usage.
  2. Use freshly prepared and chilled CCMB.
  3. Prechill the centrifuge before usage.
  4. Perform pellet resuspension gently.


Transformation


It is defined as the process by which exogenous or foreign DNA is introduced into competent E.coli cells. Recombinant DNA containing genes coding for specific proteins are generally transformed into these competent cells.


Transformation of Competent Cells


  1. Thaw and spin down the plasmid.
  2. Combine the contents of the microfuge tubes.
  3. Add 2µl of the required plasmid to the vials.
  4. Incubate the vials at 4°C for 30 minutes.
  5. Set the water bath at 42°C and maintain the temperature.
  6. Heat shock the vials at 42°C for 90 seconds.
  7. Immediately place the vials on ice and incubate them at 4°C for 30 minutes.
  8. Add 500µl of LB broth to the vials.
  9. Incubate them in a shaker at 200 rpm and 37°C for 4 hours.
  10. Plate the sample on LB agar containing Chloramphenicol or Ampicillin.


Chloramphenicol


It is a bacteriostatic, broad-spectrum antibiotic used to prevent contamination of plates. This antibiotic prevents translation by binding irreversibly onto the 50S ribosome subunit.


Chloramphenicol Preparation


  1. Retrieve the Chloramphenicol bottle from 4° C. Note that the compound is light sensitive.
  2. Add 34mg of Chloramphenicol per 1ml of ethanol in the dark.
  3. Add 1 µl of Chloramphenicol per 1ml of lukewarm LB agar.
  4. Pour approximately 20-25ml of LB agar in each plate and allow to solidify.


Ampicillin


It is an antibiotic belonging to the Penicillin group which is unique to others due to its beta-lactum ring structure. The mechanism followed by Ampicillin involves the inhibition of the 3rd and 4th step of bacterial cell wall synthesis.


Ampicillin Preparation


  1. Retrieve the Ampicillin bottle from 4° C. Note that the compound is slightly light sensitive.
  2. Add 50mg of Ampicillin per 1ml of milliQ water (ultra pure) in the dark.
  3. Add 0.5µl of Ampicillin per 1ml of lukewarm LB agar.
  4. Pour approximately 20-25ml of LB agar in each plate and allow to solidify.


CCMB Buffer for 1L


  1. 10 mM Potassium Acetate pH 7.0 (10 ml of a 1M stock/L) and autoclave it.
    • Weigh the following compounds:
    • 11.8 g of 80mM CaCl2 .2H2O.
    • 4g of 20mM MnCl2.4H2O.
    • 2g of 10mM MgCl2.6H2O.
  2. Add 100ml of 10% Glycerol.
  3. Make the volume up to 1L by adding distilled water.
  4. Adjust the pH to 6.4.
  5. Mix the contents using a magnetic stirrer for 20 minutes.
  6. Filter sterilize the buffer before use.
  7. Use prechilled buffer for experimentation.

Plasmid


An extrachromosomal, circular DNA that replicates autonomously is called a plasmid. It is usually isolated from bacteria like E.coli and can also be synthesized artificially based on molecular requirements.


Plasmid Isolation using Qiagen MidiPrep kit


Plasmid Isolation was performed using the protocol provided in the Qiagen Midi Prep kit.


Plasmid Elution


  1. Cut the gel pieces with the bands of DNA into thin slices with a sterile scalpel and add it to a microfuge tube.
  2. To the microfuge tube, add 500 µl of Binding solution.
  3. Incubate at 45 for 5 minutes.
  4. This step can be elongated to ensure complete dissolution of the agar pieces.
  5. Centrifuge at 8000 rpm for 10 seconds.
  6. Discard the supernatant and carefully resuspend the pellet with 500 µl of gel solubilizing buffer.
  7. Resuspend repeatedly until the pellets are completely dissolved.
  8. Spin the microfuge tubes at 8000 rpm for 10 seconds.
  9. Discard the supernatant and to the pellet add 500 µl of wash buffer 1
  10. Spin at 8000 rpm for 10 seconds.
  11. Discard the supernatant and allow the pellet to dry until there is no trace of wash buffer 1.
  12. Add 500 µl of wash buffer 2.
  13. Spin at 8000 rpm for 10 seconds.
  14. Discard the supernatant and allow the pellet to dry completely.
  15. Add 40 µl of TE buffer to the pellet.