Cleanup and Extraction Protocols

Ethanol Precipitation
Queen's Lysis Buffer
RNA Storage and Stabilizing Solution
DNA and RNA Extraction with the ABI6100

Ethanol Precipitation 

This protocol is ideal for 15 µl PCR and other DNA products:

  1. Add 1.5 µl 3M Sodium Acetate (for a 20 µl reaction add 2 µl, for 100 µl add 10 µl, and so on).
  2. Add 60 µl -200C 95% ethanol (for a 20 µl reaction add 40 µl, for 100 µl add 200 µl, and so on)
  3. Vortex
  4. Precipitate @ -200C for 15 minutes.
  5. Centrifuge @ 12,000 RPM for 30 minutes.
  6. Pour off supernatant
  7. Add 200 µl of -200C 70% ethanol
  8. Centrifuge @ 12,000 (or max) RPM for 10 minutes
  9. Pipette off the supernatant, being careful not to scape the bottom of the tube but effectively drawing off the ethanol. A white pellet is usually noticeable at the bottom. You should not see a pool of ethanol in the boot of the tube, otherwise the sample will not dry properly. 
  10. Air dry pellet at room temperature (approx. 30 min), preferably under a fumehood.
  11. Resuspend in 20 µl 10mM Tris (pH 8.5) or ddH2O
  12. Check the concentration of your sample with the Nanodrop or on a 1% agarose gel using a concentration ladder.
Queen's Lysis Buffer 1X

0.01 M Tris
0.01 M NaCl
0.01 M disodium-EDTA
1.0% n-lauroylsarcosine
pH 8.0

To make 100ml of 10X Queens Lysis Buffer:
1.21g Tris
0.48g NaCl
3.73g K2EDTA
1g N-Lauryl sarcosyl

RNA Storage and Stabilizing Solution
Stock solutions and reagents required:
  • 0.5 M EDTA disodium, dihydrate (18.61 g/100 ml, pH to 8.0 with NaOH while stirring)
  • 1M Sodium Citrate trisodium salt, dihydrate (29.4 g/100 ml, stir to dissolve)
  • Ammonium Sulfate, powdered; Sterile water.
Combine:
  • 40 ml 0.5 M EDTA
  • 25 ml 1M Sodium Citrate
  • 700 g Ammonium Sulfate
  • 935 ml of sterile distilled water

Stir ingredients on a hot plate under low heat until the Ammonium Sulfate is completely dissolved. Allow to cool, adjust the pH of the solution to pH5.2 using 1M H2SO4. Store either at room temperature or refrigerated.

DNA and RNA Extraction with the ABI6100​

An ABI6100 is located in the Gel Prep Lab next to the Genetics Facility. Please contact us if you would like to use it. The following document outlines protcols for isolating RNA or Genomic DNA using the ABI6100:

ABI6100