Protocol/1

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Protocol 1: Transformations
 
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What you will need:
 
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* Competent DH5α cells (50 uL per transformation)
 
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* 2 uL of plasmid you want to transform
 
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* 800 uL of LB (non-contaminated) per transformation
 
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* Plate with correct antibiotics
 
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Procedure:
 
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* Chill a box of pipette tips at -4 C and put your reaction tubes on ice.
 
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* Obtain DH5α cells from the –80 C freezer and thaw in palm. Just as they thaw, store on ice for 10 minutes.
 
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* Use a chilled tip to transfer 50 ul of the DH5α cells into each tube. Set aside one tube as a control.
 
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* Pipet 2uL of the plasmid (up to 25ng per 50 ul of competent cells, plasmid amt. should not exceed 5% that of the competent cells) into each tube. Swirl.
 
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* Store on ice for 30 minutes.
 
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* Place tubes in incubator set to 42 C for EXACTLY 90s.
 
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* Return the tubes to ice for 2 min.
 
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* Add 800uL of LB to each tube.
 
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* Incubate at 37 C for 45 min, ensure that the tube has at least 2ml capacity, to properly aerate the cells.
 
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* Spread cells on plate with appropriate antibiotic: plate between 50- 300ul each. Let dry.
 
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* Place plates inverted in incubator at 37 C for 12-16 hours.
 
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[[Team:Alberta/Notebook/protocols| Back]]
 
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Latest revision as of 17:17, 26 October 2010