Team:Hawaii/Make Competent E. Coli

Overview
This protocol is a variant of the Hanahan protocol Hanahan91 using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the [[Media:pat6855494.pdf | Bloom05 patent]] as well. This protocol has been tested on TOP10, MachI and BL21(DE3) cells. See Bacterial Transformation for a more general discussion of other techniques. The [[Media:pat6960464.pdf | Jesse '464 patent]] describes using this buffer for DH5&alpha; cells. The [[Media:pat6709852.pdf | Bloom04]] patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells.

'''This is the chemical transformation protocol used by Tom Knight and the Registry of Standard Biological Parts.

Materials

 * Detergent-free, sterile glassware and plasticware (see procedure)
 * Table-top OD600nm spectrophotometer
 * SOB

CCMB80 buffer

 * 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)
 * 80 mM CaCl2.2H2O (11.8 g/L)
 * 20 mM MnCl2.4H2O (4.0 g/L)
 * 10 mM MgCl2.6H2O (2.0 g/L)
 * 10% glycerol (100 ml/L)
 * adjust pH DOWN to 6.4 with 0.1N HCl if necessary
 * adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
 * sterile filter and store at 4&deg;C
 * slight dark precipitate appears not to affect its function

Eliminating detergent
Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic must be detergent free for these protocols. The easiest way to do this is to avoid washing glassware, and simply rinse it out. Autoclaving glassware filled 3/4 with DI water is an effective way to remove most detergent residue. Media and buffers should be prepared in detergent free glassware and cultures grown up in detergent free glassware.

Prechill plasticware and glassware
Prechill 250mL centrifuge tubes and screw cap tubes before use.

Preparing seed stocks

 * Streak TOP10 cells on an SOB plate and grow for single colonies at 23&deg;C
 * room temperature works well
 * Pick single colonies into 2 ml of SOB medium and shake overnight at 23&deg;C
 * room temperature works well
 * Add glycerol to 15%
 * Aliquot 1 ml samples to Nunc cryotubes
 * Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes
 * This step may not be necessary
 * Place in -80&deg;C freezer indefinitely.

Preparing competent cells

 * Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20&deg;C to an OD600nm of 0.3
 * This takes approximately 16 hours.
 * Controlling the temperature makes this a more reproducible process, but is not essential.
 * Room temperature will work. You can adjust this temperature somewhat to fit your schedule
 * Aim for lower, not higher OD if you can't hit this mark
 * Centrifuge at 3000g at 4&deg;C for 10 minutes in a flat bottom centrifuge bottle.
 * Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
 * It is often easier to resuspend pellets by mixing before adding large amounts of buffer
 * Gently resuspend in 80 ml of ice cold CCMB80 buffer
 * sometimes this is less than completely gentle. It still works.
 * Incubate on ice 20 minutes
 * Centrifuge again at 4&deg;C and resuspend in 10 ml of ice cold CCMB80 buffer.
 * Test OD of a mixture of 200 &mu;l SOC and 50 &mu;l of the resuspended cells.
 * Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
 * Incubate on ice for 20 minutes
 * Aliquot to chilled screw top 2 ml vials or 50 &mu;l into chilled microtiter plates
 * Store at -80&deg;C indefinitely.
 * Flash freezing does not appear to be necessary
 * Test competence (see below)
 * Good cells should yield around 100 - 400 colonies
 * Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles.

Measurement of competence

 * Transform 50 &mu;l of cells with 1 &mu;l of standard pUC19 plasmid (Invitrogen)
 * This is at 10 pg/&mu;l or 10-5 &mu;g
 * Hold on ice 0.5 hours
 * Heat shock 60 sec at 42C
 * Add 250 &mu;l SOC
 * Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
 * using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
 * For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
 * Ampicillin and kanamycin appear to do fine with 1 hour growth
 * Plate 20 &mu;l on AMP plates using 3.5 mm glass beads
 * Transformation efficiency is 15 x colony count x 105

5x Ligation Adjustment Buffer

 * Intended to be mixed with ligation reactions to adjust buffer composition to be near the CCMB80 buffer
 * KOAc 40 mM  (40 ml/liter of 1 M KOAc solution, pH 7.0)
 * CaCl2 400 mM (200 ml/l of a 2 M solution)
 * MnCl2 100 mM (100 ml/l of a 1 M solution)
 * Glycerol 46.8% (468 ml/liter)
 * pH adjustment with 2.3% of a 10% acetic acid solution (12.8ml/liter)
 * Previous protocol indicated amount of acetic acid added should be 23 ml/liter but that amount was found to be 2X too much per tests on 1.23.07 --Meaganl 15:50, 25 January 2007 (EST)
 * water to 1 liter
 * autoclave or sterile filter
 * Test pH adjustment by mixing 4 parts ligation buffer + 1 part 5x ligation adjustment buffer and checking pH to be 6.3 - 6.5


 * Reshma 10:49, 11 February 2008 (CST): Use of the ligation adjustment buffer is optional.

An Additional Protocol for Making Competent Cells
Making Competent Cells: Rubidium Chloride Method

From Dr. Callahan’s Lab

1.)Inoculate 2 ml of growth medium with single colony of recipient strain from fresh plate Incubate over night at 37º C Inoculate 100 ml of growth medium with 1 ml of over night culture Incubate at 37º C till OD550=0.48 (approximately 2-2.5 hours)

2.) Making competent cells (Keep cells on ice all the time!!!!!)
 * Incubate flask of cells for 10 minutes on ice
 * Centrifugate cells at 4º C and 5000 rpm in SS-34 rotor or equivalent for 5 minutes
 * Remove supernatant completely, keep cells on ice
 * Resuspend the cells in 15 ml ice-cold TJB1
 * Icubate suspension 5 minutes on ice
 * Centrifuge cells at 4º C and 5000 rpm for 5 minutes
 * Remove the supernatant
 * Resuspend pellet in 4 ml icecold TJB 2
 * Aliquote the cell suspension in 50 ul amounts in ice-cold 1.5 ml Eppendorf tubes
 * Freeze them fast at -80º C (EtOH bath in –80 freezer works well)
 * Check the transformation efficiency with one tube of competent cells

Buffers: 100 mM RbCl 3.0 g 6.0 g 50 mM MnCl2 x 4 H2O 2.47 g 4.94 g 30mM K-acetate 0.74 g 1.48 g 10 mM CaCl2 x 2H2O 0.36 g 0.72 g 15% glycerol 37.5 ml 75 ml - adjust pH to 5.8 (with 0.1M HCl, only a little bit), filter sterilize, store at 4ºC. 10 mM MOPS 0.20 g 0.40 g 0.50 g 10 mM RbCl 0.12 g 0.24 g 0.30 g 75 mM CaCl2 x 2 H2O 1.10 g 2.20 g 2.75 g 15% glycerol 15 ml 30 ml 37.5 ml - adjust pH to 7.0 (with NaOH), filter sterilize, store at 4º C
 * TJB 1: 250 ml 500 ml
 * TJB 2: 100 ml 200 ml 250 ml