User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/22: Difference between revisions

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==Entry title==
==Transformations==
* Insert content here...
# Place plastic culture tubes on ice for about 15 min.
# Place DNA for transformation on ice.
# After DNA has thawed, place cells on ice. (Note - want to minimize the time that the cells are out of freezer and on ice).
# Add 1, 2, or 3uL of DNA to the appropriately labeled culture tube and let sit in ice for 1 minute.
# Add cells (30uL, 40uL, or 50uL) to the tube on top of the DNA and gently pipette up and down to mix cells and DNA.
# Put tube back in ice for 4 minutes.
# Heat shock at 42°C for 80 seconds.
# Add 100uL of SOC media.
# Shake at 37°C for 1 hour.
# Plate 50uL of culture media on LB/amp plates (prepared yesterday: 6.25g LB (w/o NaCl) + 2.5g NaCl + 5g agar + 250mL dH<sub>2</sub>O, autoclave liquid cycle, cool to about 60°C, add 250μL 100mg/mL amicillin, pour plate, store at 4°C).
# Incubate inverted overnight at 37°C.




Revision as of 11:31, 21 June 2012

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Transformations

  1. Place plastic culture tubes on ice for about 15 min.
  2. Place DNA for transformation on ice.
  3. After DNA has thawed, place cells on ice. (Note - want to minimize the time that the cells are out of freezer and on ice).
  4. Add 1, 2, or 3uL of DNA to the appropriately labeled culture tube and let sit in ice for 1 minute.
  5. Add cells (30uL, 40uL, or 50uL) to the tube on top of the DNA and gently pipette up and down to mix cells and DNA.
  6. Put tube back in ice for 4 minutes.
  7. Heat shock at 42°C for 80 seconds.
  8. Add 100uL of SOC media.
  9. Shake at 37°C for 1 hour.
  10. Plate 50uL of culture media on LB/amp plates (prepared yesterday: 6.25g LB (w/o NaCl) + 2.5g NaCl + 5g agar + 250mL dH2O, autoclave liquid cycle, cool to about 60°C, add 250μL 100mg/mL amicillin, pour plate, store at 4°C).
  11. Incubate inverted overnight at 37°C.



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