Team:Groningen/protocols/Genomic DNA extraction

From 2013.igem.org

(Difference between revisions)
Line 32: Line 32:
<li>isopropanol RT</li>
<li>isopropanol RT</li>
<li>70% ethanol RT</li>
<li>70% ethanol RT</li>
-
<li>BBA+lysozymE+RNAse (Birnboim solution A chromosomal DNA isolation + 5mg/ml lysozyme + 3 &micro;l RNAse (pinch of RNAse)</li>
+
<li>BBA + lysozymE + RNAse (Birnboim solution A chromosomal DNA isolation + 5 mg/ml lysozyme + 3 &micro;l RNAse (pinch of RNAse)</li>
<li>Promega Nuclei Lysis Solution CAT# A7943</li>
<li>Promega Nuclei Lysis Solution CAT# A7943</li>
<li>Promega Precipitation Solution CAT# A7953</li>
<li>Promega Precipitation Solution CAT# A7953</li>

Revision as of 15:34, 27 September 2013

Genomic DNA extraction

Materials:
  • 1.5 ml microcentrifuge tubes
  • 2 ml microcentrifuge tubes
  • heatblock, 80°C
  • stove, 65°C
  • heatblock, 55°C
  • isopropanol RT
  • 70% ethanol RT
  • BBA + lysozymE + RNAse (Birnboim solution A chromosomal DNA isolation + 5 mg/ml lysozyme + 3 µl RNAse (pinch of RNAse)
  • Promega Nuclei Lysis Solution CAT# A7943
  • Promega Precipitation Solution CAT# A7953

Reaction procedure:
  1. Add 2-4 ml of an overnight culture to a 1.5/2 ml microcentrifuge tube.
  2. Centrifuge at 14000g for 1 minute to pellet the cells. Remove the supernatant.
  3. Resuspend the cells thoroughly in 300μl BBA+lysozyme+RNase.
  4. Incubate the sample at 55°C for 10 minutes.
  5. Add 500 μl of Nuclei Lysis Solution. The suspension should be clear.
  6. Incubate at 80°C for 5 minutes to lyse the cells; then cool to room temperature.
  7. Add 200 μl of Protein Precipitation Solution to the RNase-treated cell lysate. Vortex vigorously at high speed for 20 seconds to mix the Protein Precipitation Solution with the cell lysate. The suspension should be ‘milky’ colored.
  8. Incubate at ice for 10 minutes.
  9. Centrifuge at 14000g for 10 minutes.
  10. Transfer the supernatant containing the DNA to a clean 1.5/2 ml microcentrifuge tube containing 600/800 μl isopropanol. Fill it up to 1.5/2 ml with isopropanol.
  11. Gently mix by inversion until thread-like strands of DNA form a visible mass.
  12. Centrifuge at 14000g for 10 minutes.
  13. Carefully pour off the supernatant and drain the tube on clean absorbent paper. Add 600 μl of room temperature 70% ethanol and gently invert the tube several times to wash the DNA pellet.
  14. Centrifuge at 14000g for 5 minutes. Carefully aspirate the ethanol.
  15. Drain the tube on clean absorbent paper and allow the pellet to air-dry for 10-15 minutes (stove at 45°C).
  16. Add 100 μl TE buffer or water (Elution Buffer from the Roche Purification Kit) to the tube and rehydrate the DNA by incubating at 65°C for 15 minutes. Periodically mix the solution by gently tapping the tube.
  17. Transfer the buffer + DNA to a 1.5 ml microcentrifuge tube. Store the DNA at 2-8°C.