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| == Preparation of competent E. Coli (chemical method) ==
| | The Biolab SOPs are now stored on github; |
| {| class="wikitable"
| | https://github.com/london-biohackers/biohackspace-sops/blob/master/index.rst |
| |Author: S. Thompson
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| |Approved by:
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| |SOP No. LBL07001
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| |-
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| |Signed:
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| |Signed:
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| |Effective from:
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| |-
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| |Date:
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| |Date:
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| |Last edited:
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| |}
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| '''1. Purpose'''
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| This describes the procedure for preparing aliquots of competent E. Coli lab strains such as DH5alpha for subsequent transformation.
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| '''2. Scope'''
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| This should be observed for all such E. Coli preparation within the LBL lab but does not include variations for other species or preparations for other transformation methods e.g electroporation.
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| '''3. Responsibilities'''
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| The operator performing the transformation is responsible for their own safety and that of others in the vicinity during the procedure. | |
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| '''4. Materials'''
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| E. Coli. (plate colony or glycerol stock)<br>
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| Ice bucket.<br>
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| 0.1M CaCl.<br>
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| 0.1M MgCl.<br>
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| 50mL centrifuge tubes.<br>
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| 15mL centrifuge tubes.<br>
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| 1.5-2.0mL eppendorf tubes.<br>
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| LB broth (100mL in sterile erhlenmeyer flask)<br>
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| Incubator (shaking) 37C <br>
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| '''5. Related documents'''
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| LB broth preparation<br>
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| LB Agar selection media preparation<br>
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| E. coli heat shock tranformation<br>
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| Rapid transformation<br>
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| '''6. Definitions'''
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| ...
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| '''7. Procedures'''
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| Note: all procedures to be performed under aseptic conditions.
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| '''7.1''' Innoculate 5mL LB in 15mL tubes and incubate overnight (or until cloudy) at 37C.<br>
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| '''7.2''' Innoculate 100mL LB in conical flask using 2mL over the overnight culture.<br>
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| '''7.3''' Incubate at 37C (preferably with shaking) until an O.D600 of 0.2 to 0.4 is reached (without shaking incubator or if inoculating from a colony this may longer to achieve).<br>
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| '''7.4''' Incubate on ice for 30 minutes (from this point on care sure be taken to keep all samples on ice at every step).<br>
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| '''7.5''' Divide amongst 50mL tubes and centrifuge at 5000rpm for 10 mins to pellet the cells.<br>
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| '''7.6''' Decant supernatant and resuspend by pipeting with 5mL ice cold 0.1M CaCl2.<br>
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| '''7.7''' Incubate on ice for 20 mins.<br>
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| '''7.8''' Spin down again and remove supernatant.<br>
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| '''7.9''' Resuspend by pipeting with 5mL ice cold 0.1M MgCl2 (this step can be skipped or replaced with CaCl2).<br>
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| '''7.10''' Incubate on ice for 20 mins.<br>
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| '''7.11''' Spin down and resuspend with 0.1M CaCl2 (with 15% glycerol if storing aliquots frozen).<br>
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| '''7.12''' Snap freeze aliquots with EtOH and dry ice, or LN2, or proceed directly to heat shock transformation protocol.<br>
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| '''7.13''' Competent aliquots can stored frozen at -80 (storage at -20 has not yet been validated).<br>
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| '''8. Resources'''
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| ...
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| [[Category:Biohacking]]
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