TOP10 chemically competent cells

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This protocol is a variant of the Hanahan protocol using CCMB80 buffer for DH10B and TOP10 strains.  It builds on Example 2 of the  [[Media:pat6855494.pdf | Bloom05 patent]] as well.
This protocol is a variant of the Hanahan protocol using CCMB80 buffer for DH10B and TOP10 strains.  It builds on Example 2 of the  [[Media:pat6855494.pdf | Bloom05 patent]] as well.
 +
 +
===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.
===Preparing seed stocks===
===Preparing seed stocks===
-
* streak TOP10 cells on an [[SOB]] (no Mg) plate and grow for single colonies at 23 C
+
* streak TOP10 cells on an [[SOB]] plate and grow for single colonies at 23 C
 +
** room temperature works well
* Pick single colonies into 2 ml of SOB medium  and shake overnight at 23 C
* Pick single colonies into 2 ml of SOB medium  and shake overnight at 23 C
 +
** room temperature works well
* Add glycerol to 15%
* Add glycerol to 15%
* Aliquot 1 ml samples to Nunc cryotubes
* 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
* 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 freezer indefinitely.
* Place in -80 freezer indefinitely.
===Preparing competent cells===
===Preparing competent cells===
-
* Inoculate 250 ml of [[SOB]] medium with 1 ml vial of seed stock and grow at 23 C to an OD of 0.3
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* Inoculate 250 ml of [[SOB]] medium with 1 ml vial of seed stock and grow at 20 C to an OD of 0.5
 +
** This takes approximately 16 hours.  Controlling the temperature makes this a more reproducible process, but is not essential.  Room temperature will work.
* Centrifuge at 3000g / 4C for 10 minutes in a flat bottom centrifuge bottle.
* Centrifuge at 3000g / 4C for 10 minutes in a flat bottom centrifuge bottle.
-
* Resuspend in 80 ml of ice cold CCMB80 buffer
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** Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
 +
* Gently resuspend in 80 ml of ice cold CCMB80 buffer
* Incubate on ice 20 minutes
* Incubate on ice 20 minutes
* Centrifuge again at 4C and resuspend in 20 ml of ice cold CCMB80 buffer.
* Centrifuge again at 4C and resuspend in 20 ml of ice cold CCMB80 buffer.
* Incubate on ice for 20 minutes
* Incubate on ice for 20 minutes
-
* Aliquot to chilled screw top 2 ml vials
+
* Aliquot to chilled screw top 2 ml vials or 50 ul into chilled microtiter plates
* Store at -80C indefinitely.
* Store at -80C indefinitely.
 +
** Flash freezing does not appear to be necessary
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* 10% glycerol (100 ml/l)
* 10% glycerol (100 ml/l)
* adjust pH to 6.4 with 0.1N HCl
* adjust pH to 6.4 with 0.1N HCl
-
 
+
** adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
-
 
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* sterile filter and store at 4C.
-
15/10 medium:
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-
 
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-
* 1.5% yeast extract
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-
* 1% Bacto-Tryptone
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-
* 10mM NaCl
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-
* 2mM KCl
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-
* 10 mM MgCl<sub>2</sub>
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-
* 10 mM MgSO<sub>4</sub>
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-
 
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Experimental results:
Experimental results:
* Transform 50 &mu;l of cells with 1 &mu;l of standard pUC19 plasmid (Invitrogen)
* Transform 50 &mu;l of cells with 1 &mu;l of standard pUC19 plasmid (Invitrogen)
-
* Hold on ice 1.5 hours
+
* Hold on ice 0.5 hours
-
* Heat shock 45 sec
+
* Heat shock 60 sec
* Add 250 &mu;l SOC
* Add 250 &mu;l SOC
-
* Incubate at 37 C for 1 hour
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* Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
-
* Plate 20 &mu;l on AMP plates
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* Plate 20 &mu;l on AMP plates using 3.5 mm glass beads
Colony count was 420 on experimental plate.  This is 420*15 = 6300 transforms/10 pg or 6.3 x 10<sup>8</sup> transforms/&mu;g.  The control from Invitrogen was 210 colonies under identical conditions, or 3.2 x 10<sup>8</sup> transforms/&mu;g
Colony count was 420 on experimental plate.  This is 420*15 = 6300 transforms/10 pg or 6.3 x 10<sup>8</sup> transforms/&mu;g.  The control from Invitrogen was 210 colonies under identical conditions, or 3.2 x 10<sup>8</sup> transforms/&mu;g
 +
 +
6/7/06 results of transformations:

Revision as of 13:07, 7 June 2006

This protocol is a variant of the Hanahan protocol using CCMB80 buffer for DH10B and TOP10 strains. It builds on Example 2 of the Bloom05 patent as well.

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.

Preparing seed stocks

  • streak TOP10 cells on an SOB plate and grow for single colonies at 23 C
    • room temperature works well
  • Pick single colonies into 2 ml of SOB medium and shake overnight at 23 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 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 OD of 0.5
    • This takes approximately 16 hours. Controlling the temperature makes this a more reproducible process, but is not essential. Room temperature will work.
  • Centrifuge at 3000g / 4C for 10 minutes in a flat bottom centrifuge bottle.
    • Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
  • Gently resuspend in 80 ml of ice cold CCMB80 buffer
  • Incubate on ice 20 minutes
  • Centrifuge again at 4C and resuspend in 20 ml of ice cold CCMB80 buffer.
  • Incubate on ice for 20 minutes
  • Aliquot to chilled screw top 2 ml vials or 50 ul into chilled microtiter plates
  • Store at -80C indefinitely.
    • Flash freezing does not appear to be necessary


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 to 6.4 with 0.1N HCl
    • adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
  • sterile filter and store at 4C.


Experimental results:

  • Transform 50 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen)
  • Hold on ice 0.5 hours
  • Heat shock 60 sec
  • Add 250 μl SOC
  • Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
  • Plate 20 μl on AMP plates using 3.5 mm glass beads

Colony count was 420 on experimental plate. This is 420*15 = 6300 transforms/10 pg or 6.3 x 108 transforms/μg. The control from Invitrogen was 210 colonies under identical conditions, or 3.2 x 108 transforms/μg

6/7/06 results of transformations:

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