Duke University/24 June 2009

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Ran gel to check PCR
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[[Team:Duke | Return]]
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==June 24, 2009==
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*E: Gel to check PCR
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*R: Correct band (faint)
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*C: Do transformation with competent cells
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Transformation with High Efficiency GC5 Competent Cells
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# Remove competent cells from -70°C and place on ice. Thaw for 5-10 min.
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# Gently mix cells by tapping tube.
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# Add 1-50 ng DNA (1 ul control) into 50 ul cells. Swirl pipette tip while dispensing DNA. Gently tap tube to mix.
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# Place tubes on ice for 30 min.
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# Heat-shock cells for 45 sec in 42°C (water) bath. Do not shake!
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# Add 450 ul RT SOC Medium to each transformation reaction.
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# Incubate at 37°C for 1 hr with shaking at 225-250 rpm.
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# Spread on LB agar plates containing appropriate antibiotic.
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# Incubate plates at 37°C overnight (12-16 hrs).

Latest revision as of 17:27, 21 October 2009

Return

June 24, 2009

  • E: Gel to check PCR
  • R: Correct band (faint)
  • C: Do transformation with competent cells

Transformation with High Efficiency GC5 Competent Cells

  1. Remove competent cells from -70°C and place on ice. Thaw for 5-10 min.
  2. Gently mix cells by tapping tube.
  3. Add 1-50 ng DNA (1 ul control) into 50 ul cells. Swirl pipette tip while dispensing DNA. Gently tap tube to mix.
  4. Place tubes on ice for 30 min.
  5. Heat-shock cells for 45 sec in 42°C (water) bath. Do not shake!
  6. Add 450 ul RT SOC Medium to each transformation reaction.
  7. Incubate at 37°C for 1 hr with shaking at 225-250 rpm.
  8. Spread on LB agar plates containing appropriate antibiotic.
  9. Incubate plates at 37°C overnight (12-16 hrs).
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