Quick 'n' Easy Competent E. coli

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(Generating Competent E. coli)
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*Once this OD is reached, place the flask in an ice bath for 15 minutes with gentle shaking. (Tip: adding some water to the ice bucket can help to ensure that the flask is rapidly and evenly cooled)
 
*Once this OD is reached, place the flask in an ice bath for 15 minutes with gentle shaking. (Tip: adding some water to the ice bucket can help to ensure that the flask is rapidly and evenly cooled)
 
**Place centrifuge tube(s) and TSS buffer in the ice bath as well
 
**Place centrifuge tube(s) and TSS buffer in the ice bath as well
*Centrifuge bacteria for 10 mins/4000 RPM/4<sup>o</sup>C
+
*Centrifuge bacteria for 10 mins/4000xg/4<sup>o</sup>C
 
*Remove supernatant and re-suspend pellet in 10 mL TSS buffer
 
*Remove supernatant and re-suspend pellet in 10 mL TSS buffer
 
*Aliquot 50-200 μL of bacterial suspension into 1.7 mL micro-centrifuge tubes and snap-freeze in dry ice or liquid nitrogen, before storing in -80<sup>o</sup>C freezer
 
*Aliquot 50-200 μL of bacterial suspension into 1.7 mL micro-centrifuge tubes and snap-freeze in dry ice or liquid nitrogen, before storing in -80<sup>o</sup>C freezer
 
**Snap-freezing tubes has been shown to increase transformation efficiency by up to 20x! If LN<sub>2</sub> and dry ice are both unavailable, place tubes on ice and move to -80<sup>o</sup>C as soon as possible
 
**Snap-freezing tubes has been shown to increase transformation efficiency by up to 20x! If LN<sub>2</sub> and dry ice are both unavailable, place tubes on ice and move to -80<sup>o</sup>C as soon as possible
 
  
 
== Notes on Transformation with TSS-Competent E. coli ==
 
== Notes on Transformation with TSS-Competent E. coli ==

Revision as of 23:11, 17 July 2019

This protocol can be used to generate competent E. coli of the following strains:

  • DH5a
  • DH1
  • JM109

This will NOT work for BL-21 nor ZYCY10P3S2T (minicircle) strains


TSS Buffer Recipe

Note: TSS Buffer is used at 1/10th culture volume, e.g. 100 mL culture is re-suspended in 10 mL TSS

  • In a 15 mL conical tube, add:
    • 1.0 g PEG-8000
    • 300 μL 1M MgCl2 stock
    • 9 mL LB Broth (optionally sterile)
  • Warm in 55oC water bath to dissolve PEG
  • Filter sterilize through a 0.22μm filter into a new (sterile) 15 mL conical tube
  • Using sterile technique, add 500 μL DMSO and vortex to mix
    • IMPORTANT: do not add DMSO before filtration as it will destroy our cellulose filters
  • Chill to 4oC before use


Generating Competent E. coli

The night before prep:

  • Inoculate 2-5 mL Super Optimal Broth (SOB) with DH5a from a glycerol stock and grow overnight at 37oC on shaker at 200 RPM
  • Prepare 100 mL of LB broth (or custom amount, adjust TSS volume accordingly)

Day of prep:

  • Use LB broth to blank spectrophotometer at OD600
  • Add entire overnight culture to flask and swirl vigorously
  • Record starting OD of flask
  • Incubate at 37oC on shaker at 150-200 RPM until OD600 reaches 0.3-0.5, optimally 0.45. DO NOT OVERGROW!!
    • To avoid missing this, check OD every 30-60 minutes until OD > 0.2 -- at which point, check every 10-15 minutes
  • Once this OD is reached, place the flask in an ice bath for 15 minutes with gentle shaking. (Tip: adding some water to the ice bucket can help to ensure that the flask is rapidly and evenly cooled)
    • Place centrifuge tube(s) and TSS buffer in the ice bath as well
  • Centrifuge bacteria for 10 mins/4000xg/4oC
  • Remove supernatant and re-suspend pellet in 10 mL TSS buffer
  • Aliquot 50-200 μL of bacterial suspension into 1.7 mL micro-centrifuge tubes and snap-freeze in dry ice or liquid nitrogen, before storing in -80oC freezer
    • Snap-freezing tubes has been shown to increase transformation efficiency by up to 20x! If LN2 and dry ice are both unavailable, place tubes on ice and move to -80oC as soon as possible

Notes on Transformation with TSS-Competent E. coli

Incubate thawed aliquot of competent E. coli with appropriate amount of DNA (≤20 μL in a 100 μL aliquot) for at least 5-10 minutes (optimally 30 minutes). The 42oC heat-shock step is not required when working with TSS-competent E. coli, though it does increase transformation efficiency by 2-5x. TSS-Competent cells respond best to 30-45 second heat-shock, not any longer. It is recommended to incubate for as long as possible (up to 30 minutes) when not performing a heat-shock step. Recover for 45-75 minutes in Super Optimal Broth (SOB), then plate or inoculate directly from transformation reaction.

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