Lidstrom:Chemical Transformation

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(New page: Back to Protocols You can chose between chemically competent cells and electrocompetent cells. Andrew & Nicole make chemically competent cells for the lab to use. ...)
Current revision (20:19, 14 October 2013) (view source)
 
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*Plate 50-100 uL cells on LB (+antibiotic(s)) agar plate
*Plate 50-100 uL cells on LB (+antibiotic(s)) agar plate
**If you are worried about having a lawn, do one plate with more cells and dilute a fraction of the cells and plate a diluted aliquot.
**If you are worried about having a lawn, do one plate with more cells and dilute a fraction of the cells and plate a diluted aliquot.
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== Note: electroporation is much more efficient than chemical transformation ==
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* Transformations done with 1uL of 0.1 ng/uL DNA (pGA 3K3 RFP from Rob Egbert).  0.1 ng is ~10^8 plasmid copies.  Tami's electrocomp cells yielded several hundred colonies, which suggests the transformation efficiency is on the order of 100/10^8 = 0.000001 = 0.0001% efficiency.  The chemically competent cells yielded < 10 colonies, which is much lower efficiency.
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* [[image:2013_10_14_comparison_of_electroporation_and_chemical_transformation.jpg|thumb|center|upright=2.0|Top10 cells prepared for electroporation and chemical transformation.  Both were transformed with ~10^8 copies of DNA (0.1 ng of plasmid.)]]

Current revision

Back to Protocols

You can chose between chemically competent cells and electrocompetent cells. Andrew & Nicole make chemically competent cells for the lab to use. Several strains are kept in stock in the -80oC freezer.

Using Chemically Competent Cells

  • Thaw frozen (-80oC) competent cells on ice.
    • You REALLY dont want it to get too warm; add plasmids while it is still slushy.
  • Add 1-10 uL DNA
    • 10 uL if ligated plasmid. Use only 1 uL if regular plasmid from miniprep
  • Incubate @ 42oC for 45 sec - 1 min
  • Incubate on ice for 2 min
  • Add 1 mL LB
    • Sandy, Ceci, & Janet use 500 uL
  • Incubate at 37oC for 45 min - 1 hr in eppendorf tubes
    • Ideally shaking though it may not matter. You can tape your tubes to a rack in the shaker. Tape them well if you do -- they fly off!
  • Pellet cells by centrifugation
    • keep the ~100 uL droplet after you pour it off (Andrew)
  • Plate 50-100 uL cells on LB (+antibiotic(s)) agar plate
    • If you are worried about having a lawn, do one plate with more cells and dilute a fraction of the cells and plate a diluted aliquot.

Note: electroporation is much more efficient than chemical transformation

  • Transformations done with 1uL of 0.1 ng/uL DNA (pGA 3K3 RFP from Rob Egbert). 0.1 ng is ~10^8 plasmid copies. Tami's electrocomp cells yielded several hundred colonies, which suggests the transformation efficiency is on the order of 100/10^8 = 0.000001 = 0.0001% efficiency. The chemically competent cells yielded < 10 colonies, which is much lower efficiency.
  • Top10 cells prepared for electroporation and chemical transformation.  Both were transformed with ~10^8 copies of DNA (0.1 ng of plasmid.)
    Top10 cells prepared for electroporation and chemical transformation. Both were transformed with ~10^8 copies of DNA (0.1 ng of plasmid.)
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