Isolation of zebrafish genomic DNA with DNAzol reagent:

 The DNA extracted for AFLP must be clean enough for complete enzyme digestion.  We have developed the following method to rapidly prepare dirty DNA from large numbers of individual embryos while retaining aliquots that can be stored and purified more extensively at a later time point.  Dirty prep DNA can be typed with CA repeats, RAPDs or SSCPs to identify recombinant individuals.  Samples saved from the recombinants can then be purified for AFLP.

Lysis Reagents:

  1. Lysis Buffer (10 mM Tris pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.3% NP40, 0.3% Tween-20, 1.8 mg/ml proteinase K)
  2. DNAzol reagent (Gibco/BRL # 10503)
  3. 1 M Hepes
  4. 8 mM NaOH
  5. Glycogen 20mg/ml (Boehringer Manheim # 901393)


Extraction of Both Clean and Dirty Genomic DNA from the Same Samples: 
 

  1. Sort embryos or larvae and collect up to 200 animals in 5 ml glass vial.  Fix by washing twice in methanol and store in methanol at -20oC. 
  2. Put fixed animals into a petri dish to separate individuals and transfer them into wells of 96 well plates using a transfer pipette.  Remove methanol with a pulled pasteur pipette. 
  3. Dry the embryos for 10 min at 70 oC by putting the uncapped plate in a thermocycler.
  4. Add 50 µl of lysis buffer and digest overnight at 55°C in a oven.
  5. Remove debris by using a multichannel pipette set at 5 µl.
  6. Split each lysate between dirty and clean preps.  Transfer 25 µl of each lysate to a new plate and add 50 µl of DNAzol to each well.  Store the DNAzol samples at 4°C and do not freeze. 
  7. For the dirty prep portion, boil the embryos at 95°C for 10 minutes in a thermocycler and cool to 4°C.  Dilute the sample by 1:30 for PCR.  The dirty lysates can be stored at -20°C.
  8. Once recombinants have been identified the appropriate stored DNAzol lysates can be processed.  Transfer lysates to 2 ml microfuge tubes and add 1 µl of glycogen (20 µg / µl). 
  9. Precipitate DNA from the lysates by the addition of 25 µl of 100% ethanol. Mix samples by inversion, and incubate for 1 - 5 min at room temperature. 
  10. Centrifuge at 4K rpm for 2 min at RT.  Remove the supernatant while avoiding the pellet of DNA.
  11. Wash the pellet 2x with 1 ml of 95% ethanol.  Suspend the DNA in the ethanol by inverting the tube 3 times.  Respin the tubes at 4K rpm for 2 min to pellet the DNA.
  12. Air dry the DNA 15 to 30 min in an open tube.  Do not over dry DNAs.
  13. Dissolve DNA in 25 µl of 8 mM NaOH.  Pipette gently and do not vortex.  Make up 8 mM NaOH fresh each month.
  14. Neutralize DNA solution by adding 1 µl of 1 M HEPES.
  15. Use as a stock for AFLP or store at 4°C. Do not freeze.