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Technology Development Group
 
Instruments 

Hydrodynamic DNA Shearer 

Plaque Picker 

High Capacity Shaker 

Template Preparation Machine 

Flow Through Micro-Centrifuge 

Plasmid Preparation Machine 

Thermocycler 

Capillary Electrophoresis 

Oligonucleotide Synthesizer 

Arrayer 

Scanner

High Capacity Shaker 

High Capacity Shaker We 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.  

Growth Protocols 

M13 growth 

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 cassette.
  • 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.

Performance: 

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. 
 

Cell Growth

Typical parameters for cell growth are identical to M13 growth except:  
  • Growth media: TB + salts.
  • Growth Time: approx 20 hrs.
  • Strain: DH10B 
  • Oxygen Usage: < 0.5 cu. ft.
  • Oxygen Duty Cycle: 5 sec every 20 min.
  • Starting inoculum: 10,000 to 100,000 cells
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.  

Performance: 

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. 

 
High Capacity Shaker
Hi-Cap Shaker Image
Full sized Shaker Image
Hi-Cap Shaker Movie
14-Plate Cassette
14-Plate Cassette Image
Shaker Drawings
Shaker Drawings

Staff | Instruments & Protocols | Automated Sequencing System | Functional Analysis | Software Development
Stanford Genome Technology Center:
Technology Development Group
855 California Avenue | Palo Alto, CA 94304 | Phone: (650) 812-2007 | Fax: (650) 812-1975 
wwwadmin@sequence.stanford.edu