Docstoc

Genomic DNA Extraction for Microarray and NextGen Sequencing

Document Sample
Genomic DNA Extraction for Microarray and NextGen Sequencing Powered By Docstoc
					      Genomic DNA Extraction for Microarray and NextGen
                   Sequencing Analysis
                  ProtK/Phenol/PEG v0906
Notes:
Tissue homogenization is a critical point in this protocol. If you are not sure whether a
proteinase K digest will efficiently destroy the tissue, add the digestion buffer to the
tissue and use a power homogenizer (Polytron or equivalent) or a tissue grinder "Pellet
pestle" for microtubes (Sigma).

Fragmentation of DNA is not an issue for microarray or NextGen analysis. Please use
the vortex at full speed anytime indicated below.

As an example, we describe DNA isolation from mouse tails below:

  1) Remove 0.5 mm of tail into a 1.5 ml tube. The tubes must have tight-fitting caps,
     so that there are no leaks in steps below.

  2) Add 600 µl DNA digestion buffer with proteinase K added to 0.5 µg/µl final
     concentration.

DNA digestion buffer:    50 mM Tris-HCl pH 8.0
                        100 mM EDTA pH 8.0
                        100 mM NaCl
                        1% SDS

                              C
  3) Incubate overnight at 55° with shaking. Mechanical agitation greatly aids
     complete disruption of the tissue.

  4) Spin 5 min at RT, max speed to remove insoluble material. Transfer into new
     tube.

  5) Add 600 µl neutralized phenol/chloroform (1:1, pH=7.2), vortex vigorously.

   6) Spin 5 min at RT, max speed and transfer 500 µl of the upper phase to new tube.
(Use P1000 for transfer, and draw the aqueous phase gently through tip several times
after transfer if the DNA is still in large, gelatinous mass.)
  7) Add 1000 µl 100% ethanol at room temperature and shake until DNA precipitate
     forms. (approximately 1 minute). If you do not see the DNA, precipitate 30 min on
     ice.

  8) Spin 5 min at RT, max speed and carefully remove supernatant.

  9) Add 500 µl 75% ethanol and invert several times.

  10) Spin 5 min at RT, max speed and carefully remove supernatant. Air dry at room
      temperature.

                                        C
  11) Add 200 µl TE and incubate at 65 ° for 15 min to resuspend DNA. Draw DNA
                                 C
      through P200 tip after 65 ° incubation to aid in suspension.

  12) Add 2 µl RNAse A (Promega) and incubate 30 min at 37 °C.

  13) Add 200 µl neutralized phenol/chloroform (1:1, pH=7.2), vortex vigorously.

  14) Spin 5 min at RT, max speed and transfer 200 µl of the upper phase to new tube.

  15) Add 200 µl chloroform, vortex vigorously.

  16) Spin 1 min at RT, max speed and transfer 200 µl of the upper phase to new tube.

  17) Add 200 µl PEG/NaCl and mix well.

PEG 20% / NaCl 2.5M:            400 µl PEG 6000 50% (Fluka)
                                500 µl NaCl 5M
                                100 µl water

   18) Incubate 10min at 37ºC. Centrifuge for 10 min at RT, max speed.
Note: PEG pellets come in different flavors: invisible, normal pellets or clear, highly
viscous solutions with a turbid layer on top.

  19) Immediately remove supernatant. Add 500 µl 75% EtOH and vortex extensively.
      The pellet (if visible) should look like a "normal" pellet.

  20) Spin 5 min at RT, max speed and carefully remove supernatant. Quick spin tubes
      and remove last drop of ethanol with 10 µl tip (skip this step if you do not see the
      pellet; instead, remove EtOH drops by pipetting). Air dry 5 min at RT.

  21) Add 50 µl water and incubate over night at 4 °C.

  22) Dissolve next morning by pipetting and quantify by Nanodrop and/or Qubit.

  23) Total yield is approximately 10 µg DNA.

				
DOCUMENT INFO