Document1 9-fev-16
Top10 chemically competent cells (CCMB80 protocol)
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Grow culture from fresh preinoculum/1:100 overnight preinoculum in 100 to 200
ml SOB to 0,3-0,4 OD600 at room temperature - the given quantities are for 200
ml cultures (this protocol worked so far with DH10B, SURE, XL1blue and BL21
cells, others possibly work as well)
Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle or 2-4
new sterile pre-chilled 50 ml falcon tubes.
o Flat bottom centrifuge tubes make the fragile cells much easier to
resuspend
o It is often easier to resuspend pellets by mixing before adding large
amounts of buffer
Gently resuspend in 60 ml (200 ml start volume) of ice cold CCMB80 buffer
o sometimes this is less than completely gentle. It still works.
Incubate on ice for 20 minutes
Centrifuge again at 4°C and resuspend in 4 ml of ice cold CCMB80 buffer.
Test OD of a mixture of 200 μl SOC or LB and 50 μl of the resuspended cells,
this should give OD600 nm reads of 1-1.5 in this diluted sample.
Add chilled CCMB80 to the cell suspension to yield a final OD of 5-7.5
(corresponds to 1-1.5 of the 1:5 diluted stuff)
Incubate on ice for 20 minutes
Aliquot in 200 ul aliquots to chilled 1,5 ml eppendorf vials or 50 μl into chilled
microtiter plates
Store at -80°C indefinitely.
o Flash freezing does not appear to be necessary
Test competence using quantified maxiprep-pure plasmids diluted down to 1-10
pg/µl (make this dilution fresh every time you check bacteria)
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.
CCMB80 is:
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10 mM KOAc pH 7.0 (10 ml of a 1M stock per 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
o adjusting pH up will precipitate manganese dioxide from Mn containing
solutions.
sterile filter and store at 4°C
slight dark precipitate appears not to affect its function