Protocols
Media
LB
Ingredients:
10 g Bacto-tryptone
5 g yeast extract
10 g NaCl
SOB
Ingredients
0.5% (w/v) yeast extract
2% (w/v) tryptone
10 mM NaCl
2.5 mM KCl
20 mM MgSO4
Per liter
5 g yeast extract
20 g tryptone
0.584 g NaCl
0.186 g KCl
2.4 g MgSO4
SOC
Ingredients
SOB
20 mM glucose
CCMB80 buffer for preparation of chemically competent cells
Materials
Detergent-free, sterile glassware and plasticware
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
Filter sterilize and store at 4°C
Slight dark precipitate appears not to affect its function
Procedures
Preparation of chemically competent cells
Preparing glassware and media
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°C works well. Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C.
Add glycerol to 15% and aliquot 1 ml samples into cryotubes. Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes. Place in -80°C freezer indefinitely.Preparing competent cells. Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3. This takes approximately 16 hours.
Preparing competent cells
Aim for lower, not higher OD if you can't hit this mark.Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle.Gently resuspend in 80 ml of ice cold CCMB80 buffer. Incubate on ice 20 minutes, centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer.Test OD of a mixture of 200 μl SOC and 50 μl 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 μl into chilled microtiter plates. Store at -80°C indefinitely
Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°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°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°C and resuspend in 10 ml of ice cold CCMB80 buffer.
Test OD of a mixture of 200 μl SOC and 50 μl 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 μl into chilled microtiter plates
Store at -80°C indefinitely.
Flash freezing does not appear to be necessary
Test competence (see below)
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 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen)
This is at 10 pg/μl or 10-5 μg/μl
This can be made by diluting 1 μl of NEB pUC19 plasmid (1 μg/μl, NEB part
number N3401S) into 100 ml of TE
Hold on ice 0.5 hours
Heat shock 60 sec at 42C
Add 250 μl 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 μl on AMP plates using sterile 3.5 mm glass beads
Good cells should yield around 100 - 400 colonies
Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA
We expect that the transformation efficiency should be between 5x108 and 5x109
cfu/µgDNA
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
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