Difference between revisions of "LBL07001"

Latest revision as of 20:17, 13 October 2015

The Biolab SOPs are now stored on github; https://github.com/london-biohackers/biohackspace-sops/blob/master/index.rst

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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
-|Approved by:
-|SOP No. LBL07001
-|-
-|Signed:
-|Signed:
-|Effective from:
-|-
-|Date:
-|Date:
-|Last edited:
-|}
-'''1. Purpose'''
-This describes the procedure for preparing aliquots of competent E. Coli lab strains such as DH5alpha for subsequent transformation.
-'''2. Scope'''
-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.
-'''3. Responsibilities'''
-The operator performing the transformation is responsible for their own safety and that of others in the vicinity during the procedure.
-'''4. Materials'''
-E. Coli. (plate colony or glycerol stock)<br>
-Ice bucket.<br>
-.1M CaCl.<br>
-.1M MgCl.<br>
-mL centrifuge tubes.<br>
-mL centrifuge tubes.<br>
-.5-2.0mL eppendorf tubes.<br>
-LB broth (100mL in sterile erhlenmeyer flask)<br>
-Incubator (shaking) 37C <br>
-'''5. Related documents'''
-LB broth preparation<br>
-LB Agar selection media preparation<br>
-E. coli heat shock tranformation<br>
-Rapid transformation<br>
-'''6. Definitions'''
-...
-'''7. Procedures'''
-Note: all procedures to be performed under aseptic conditions.
-'''7.1''' Innoculate 5mL LB in 15mL tubes and incubate overnight (or until cloudy) at 37C.<br>
-'''7.2''' Innoculate 100mL LB in conical flask using 2mL over the overnight culture.<br>
-'''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>
-'''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>
-'''7.5''' Divide amongst 50mL tubes and centrifuge at 5000rpm for 10 mins to pellet the cells.<br>
-'''7.6''' Decant supernatant and resuspend by pipeting with 5mL ice cold 0.1M CaCl2.<br>
-'''7.7''' Incubate on ice for 20 mins.<br>
-'''7.8''' Spin down again and remove supernatant.<br>
-'''7.9''' Resuspend by pipeting with 5mL ice cold 0.1M MgCl2 (this step can be skipped or replaced with CaCl2).<br>
-'''7.10''' Incubate on ice for 20 mins.<br>
-'''7.11''' Spin down and resuspend with 0.1M CaCl2 (with 15% glycerol if storing aliquots frozen).<br>
-'''7.12''' Snap freeze aliquots with EtOH and dry ice, or LN2, or proceed directly to heat shock transformation protocol.<br>
-'''7.13''' Competent aliquots can stored frozen at -80 (storage at -20 has not yet been validated).<br>
-'''8. Resources'''
-...
-[[Category:Biohacking]]

Difference between revisions of "LBL07001"

Latest revision as of 20:17, 13 October 2015

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