Admin: Created page with "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 =..."

2021-02-01T19:41:57Z

Created page with "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 =..."

New page

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 as much as 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.

Quick 'n' Easy Competent E. coli - Revision history

Admin: Created page with "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 =..."

Admin: Created page with "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 =..."