Duke University/24 June 2009

From 2009.igem.org

(Difference between revisions)
 
Line 5: Line 5:
*R: Correct band (faint)
*R: Correct band (faint)
*C: Do transformation with competent cells
*C: Do transformation with competent cells
 +
 +
Transformation with High Efficiency GC5 Competent Cells
 +
# Remove competent cells from -70°C and place on ice. Thaw for 5-10 min.
 +
# Gently mix cells by tapping tube.
 +
# Add 1-50 ng DNA (1 ul control) into 50 ul cells. Swirl pipette tip while dispensing DNA. Gently tap tube to mix.
 +
# Place tubes on ice for 30 min.
 +
# Heat-shock cells for 45 sec in 42°C (water) bath. Do not shake!
 +
# Add 450 ul RT SOC Medium to each transformation reaction.
 +
# Incubate at 37°C for 1 hr with shaking at 225-250 rpm.
 +
# Spread on LB agar plates containing appropriate antibiotic.
 +
# 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).