Monday 06/15/15

Posted on June 15, 2015 by admin

10:00 am – 5:00 pm

Referee (10:00 am – 1:30 pm)

  • finished reading article
  • finished writing comments
  • summarized comments in review, summarized article contributions
  • submitted to editorial office.

Prep for RNAi experiments

Primers

  • look up primers on genomernai.org/v14/
  • order primers with t7 from IDT

Genomic DNA from Kc cells

  • protocol from Filion lab (copied from here)
  • Thanks Guillaume!

Material:

  • Extraction buffer (see below)
  • Proteinase K, 20 mg/mL stock (New England Biolabs, REF: P8102S)
  • RNase A, 1 mg/mL stock (Sigma-Aldrich, REF: R6513)
  • Chloroform
  • Isopropanol
  • 70% ethanol

extraction buffer with the following composition.

  • 0.8 M guanidine thiocyanate
  • 5 mM CaCl2
  • 20 mM EDTA
  • 5% Tween 20
  • 0.5% Triton X-100
  • 50 mM HEPES pH 5.3

Procedure:

  1. Grow Kc cells in 1.5 mL in a 6-well plate to ∼ 1-2 million cells.
  2. Transfer 1.5mL Kc cells into a 1.5 mL Eppendorf.
  3. Centrifuge at 2000 rpm for 3 minutes.
  4. Resuspend cell pellet with 500 μL extraction buffer.
  5. Add 10 μL of proteinase K and 5 μL RNase A. Invert the tube 3-5 times. Do not vortex.
  6. Incubate the sample at 55°C for 15 minutes.
  7. Add 600 μL chloroform. Emulsify by shaking. Do not vortex.
  8. Centrifuge at maximum speed at 4°C for 5 minutes.
  9. Transfer the top phase into a new 1.5 mL Eppendorf.
  10. Add 350 μL iso-propanol. Invert the tube 3-5 times. Do not vortex.
  11. Centrifuge at maximum speed at 4°C for 5 minutes.
  12. Spill the supernatant and add 70% ethanol. Invert the tube 3-5 times. Do not vortex.
  13. Centrifuge at maximum speed at 4°C for 5 minutes.
  14. Spill the supernatant. Do not let dry more than 1 minute.
  15. Dissolve DNA pellet in appropriate volume of distilled water.

Alternate

  • don’t have Guanidine thiocyanate in stock
  • Try the DNAzol approach
  • http://www.lifetechnologies.com/us/en/home/references/protocols/nucleic-acid-purification-and-analysis/dna-extraction-protocols/extraction-of-dna-using-reagent.html#proto
  • DNA seems to crash out of solution in the optional centrifugation step. Skip this step, get what seem to be good preps.
  • prepped 3 samples in 8 mM NaOH. Will test for PCR.
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