TITLE: Generating chemically competent (E. coli) cells
AUTHORS: Su Yin Phua, Nay Chi Khin, Estee Tee, Diep R. Ganguly, Kai Xun Chan, Veronica Albrecht-Borth, Barry J Pogson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for generating chemically compotent (ie. for heat shock transformation) E. coli cells for transformation (e.g. heterologous protein expression, cloning).</div></div>

[STEPS]
?. [Prepare solutions A and B]
In a beaker, prepare 250 ml of Solution A:- 100 mM MgCl2 (hexahydrate = 5.08 g; anhydrous = 2.38 g)- 10 mM MOPS (0.52g)- Up to ~250 mL H2OAdjust to pH 7 using 1 M NaOH (slowly; ~900 μL)Transfer to measuring cylinder and ensure final volume = 250 mL
?. [Prepare solutions A and B]
In a beaker, prepare 300 ml of Solution B- 10 mM MgCl2 (hexahydrate = 0.61 g; anhydrous = 0.29 g)- 10 mM MOPS (6.78g)- 50 mM CaCl2 (dihydrate = 2.21g)- Fill to ~300 ml with H2OAdjust to pH 6.5 with 1 M NaOH (~6.8 ml)Transfer to measuring cylinder and ensure final volume = 300 mL
?. [Prepare solutions A and B]
Filter sterilize (stericup) both solutions and store @ 4ºC. MOPS degrades into unknown yellow byproduct from autoclaving!
?. [Generate compotent cells]
Culture bacteria at 37ºC overnight in 5 mL LB media
?. [Generate compotent cells]
Innoculate desired of LB media using an aliquot, or entire 5 mL, overnight culture. Volumes can range from 50 - 500 mL depending on a variety of factors (e.g. lab time required, equipment available, amount of cell desired). Ensure a proper vessel is used to allow efficient aeration of cultures.e.g. Add 5 mL starter culture to 500 mL LB media (use 1-2 L connical flask) for a large batch of competent cells. Or, add 500 μl - 1 mL starter culture to 50 mL LB media (use 250 mL connical flask).Add autoclaved MgSO4 to final concentration 10 mM.
?. [Generate compotent cells]
Cool on ice for 10 mins (make sure centrifuge is refridgerated and ready)
?. [Generate compotent cells]
Pellet at 4ºC for 10 mins @ ~4200 rcf
?. [Generate compotent cells]
Remove supernatant and resuspend cells in cold solution A (volume can be adjusted to minimize the volume required for centrifugation but to adequately resuspend colonies e.g. 250 mL for 500 mL culture or 25 mL for 50 mL culture).
?. [Generate compotent cells]
Split resuspended cells equally across centrifuge bottles or falcon tubes. Incubate 10 - 20 minutes at 4ºC.
?. [Generate compotent cells]
Pellet at 4ºC for 10 mins @ ~4200 rcf
?. [Generate compotent cells]
Remove supernatant and resuspend cells in 4.3 mL of cold Solution B + 700 μL glycerol (pipette slowly).
?. [Generate compotent cells]
Make aliquots of 20, 50, and/or 200 μL (variable volumes based on plasmid to be used). Freeze in liquid N2 before storing at -80ºC.
?. [Generate compotent cells]
Remove supernatant and resuspend cells in cold solution B (repeat as per solution A).
?. [Generate compotent cells]
Incubate 30 minutes at 4ºC.
?. [Generate compotent cells]
Pellet at 4ºC for 10 mins @ ~4200 rcf
?. [Generate compotent cells]
Grow to OD600 = 0.4 - 0.5 (do not exceed 0.6). This should take between 1 - 4 hrs depending on volumne of starter culture used (ie. 5 mL starter culture ~ 1 hr to reach OD600 ~0.5).