High Capacity Shaker
have developed a high capacity, atmosphere controlled shaker for growth
of e-coli and phage in 96-well microtiter plates. The shaker holds four
14-plate cassettes ( 56 microtiter plates
max. capacity) and shakes them in a
synchronized manner to eliminate all vibrational forces and moments, which
normally would be transferred to the base by the large rotating masses.
Each cassette is housed
in a sealed tube which is periodically flushed
with 100% oxygen gas. This oxygen enrichment has boosted our M13 growth
by a factor of five over standard methods. Growth time is approximately
16 hrs and can be set internally in the shaker electronics. All M13 growth
at our center is now performed with this shaker. It has been in operation
since May 1997.
Our typical parameters for
M13 growth are as follows:
Growth media: 2XTB + salts
(salts are at same concentration as used in regular TB)
Growth Volume: 300 microliters
in each well of a COSTAR 96 well plate.
Growth Time: approx. 18 hrs
Shaking Rate: 2.5 on
our shaker (similar on Genemachines's shakers) = 520 rpm
Oxygen Usage: 26 cu. ft. per
Oxygen Injection Duty Cycle:
3 sec every 30 sec.
Temperature: 37 degrees Celsius
Starting DH12S cell concentration:
10% of OD550=1.5 overnight.
Typical phage inoculum: 1E6
phage per well.
Typical growth is approx. 2
E12 to 6 E12 pfu/ml based on inoculation with our automated plaque picker.
With optimization we have occasionally reached titers of 1 E13 pfu/ml.
Typical parameters for cell
growth are identical to M13 growth except:
Note: It is critical with cell
growth that the initial inoculum be sufficient. If the starting number
of cells is too low, the oxygen atmosphere may inhibit cell growth. If
you expect low starting cell concentrations, allow growth to proceed for
a few hours before turning on the oxygen.
Growth media: TB + salts.
Growth Time: approx 20 hrs.
Oxygen Usage: < 0.5 cu.
Oxygen Duty Cycle: 5 sec every
Starting inoculum: 10,000 to
Typical growth is approx. 8
E9 to 1 E10 cfu/ml based on starting titers of 10,000 to 100,000 cells.
THIS PERFORMANCE IS STRONGLY DEPENDENT ON INITIAL TITER AND OXYGEN LEVELS.
If growth is insufficient, try turning oxygen off entirely. You may then
slowly add oxygen on subsequent runs to determine the optimum level for
your cells and media.
The yields from both these
growth protocols tend to be three to five times greater than yields from
standard growth methods (ie. standard shakers with no oxygen enrichment).
This instrument is now available
through Genemachines. For information
call them at 650-508-1634 or e-mail them.