View source for LBL07002


== Heat shock transformation of E. coli ==
{| class="wikitable"
|Author: S. Thompson
|Approved by:
|SOP No. LBL07002
|-
|Signed:
|Signed:
|Effective from:
|-
|Date:
|Date:
|Last edited:
|}

'''1. Purpose'''

This describes the procedure for heat shock transformation in E. Coli lab strains such as DH5alpha. 

'''2. Scope'''

This should be observed for all such transformations within the LBL lab but does not include variations of other species or 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'''

Chemically competent E. Coli.<br>
Ice bucket.<br>
Water bath, 42C.<br>
Plasmid suitable for E. Coli with antibiotic selection marker<br>
LB broth (0.5 to 1mL per sample)<br>
LB Agar plates containing antibiotic matching selection marker<br>
Incubator, 37C<br>

'''5. Related documents'''

LB broth preparation<br>
LB Agar selection media preparation<br>
E. coli chemical competence<br>
Rapid transformation<br>

'''6. Definitions'''

...

'''7. Procedures'''

'''7.1''' Take frozen chemically competent aliquots and thaw on ice.<br>
'''7.2''' Add an appropriate amount of plasmid DNA (based on initial plasmid concentration). Mix carefully by pipeting or flicking the tube. <br>Note: Also remember to use a negative control such as sterile water for at least one of the samples.<br>
'''7.3''' Incubate on ice for 20 to 30 minutes.<br>
'''7.4''' Heat shock by placing the sample tubes into a 42C water bath for 60 seconds.<br>
'''7.5''' Incubate on ice again for 2 minutes.<br>
'''7.6''' Add 250 to 500ul of LB medium without antibiotic (recovery step).Incubate at 37C for 45 minutes (this can vary from 30 to 60 minutes, allow longer times for tranformations with ligation mixtures).<br>
'''7.7''' Plate onto LB agar plates containing suitable antibiotic and incubate at 37C overnight.<br>
'''7.8''' Check for colonies and compare with colony formation on negative controls.<br>



'''8. Resources'''

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[[Category:BioHacking]]