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Genotyping for Single Zebrafish (Fin Clip) or Zebrafish Embryo   

Abstract

Materials and Reagents

1. Tricane (Sigma-Aldrich, catalog number: 886-86-2 )
2. dNTPS (QIAGEN, catalog number: 201900 )
3. Proteinase K (Roche Diagnostics, catalog number: 03115836001 )
4. MEOH
5. DNA polymerase 
6. MgCl₂
7. Tris 
8. KCl
9. MgCl₂
10. Gelatine
11. BSA
12. NP40
13. Tween 20
14. PCR lysis buffer (see Recipes)
15. RAPD⁺ (see Recipes)
16. 1x RAPD buffer (see Recipes)
17. 2x RAPD⁺ buffer (see Recipes)
18. Tricane solution (see Recipes)
    

Equipment

1. Standard tabletop centrifuges
2. Incubator (55 °C and 65 °C)
3. PCR thermal cycler
4. 96-well PCR plate
5. Razors
6. Plastic plates
7. Plastic beaker
8. Forceps
   
9. Spoon
   

Procedure

1. Fin clip:
   1. Bring clean plastic plates, razors, Tricane, a plastic beaker, forceps and a spoon to fish room.
   2. Prepare a 96-well plate, with 100-200 μl of MEOH in each well. 
   3. Add 20 ml Tricane to the beaker and add clean water to dilute the tricane (1:15 dilution).
   4. Put the fish into the tricane solution and let the fish sleep. Move the fish to a clean plate and cut a small piece of the tail fin.
      
   5. Quickly put the fish back to the single box and put the tail into one well of the PCR plate with MEOH in it.
   6. Label both the single box and the well.
      
   7. Make sure the tail is in the MEOH (the fish can be kept in single boxes for up to 5 days).
      Note: A single embryo can be kept in MEOH directly.
      
      
2. Genomic DNA extraction:
   
   1. Remove as much MEOH as possible.
   2. Incubate at 55 °C for 5 min to evaporate all MEOH.
      
   3. Add 100 μl lysis buffer per well (200 μl lysis buffer for 72 hpf embryo).
   4. Incubate at 55 °C O/N and heat inactivate at 95 °C for 10 min.
      
   5. Centrifuge at 4 °C at 1,000 rpm for 2-10 min.
   6. Store DNA at -20 °C or for long time at -80 °C.
      
      
3. PCR after genomic DNA extraction
   
   1. PCR reaction set up
      X μl       RAPD+ (make up to 20 μl)
      1-2 μl    DNA
      1 μl       Forward primer (20 μM)
      1 μl       Reverse primer (20 μM)
      1 μl       Taq Polymerase
      For multiple PCRs, make a master mix (keep mixture and the plate on ice). 
   2. PCR program
      94                       1 min
      94                        30 sec
      54                        2 min
      73                        1 min
                                  go to ii. 5x
      94                        30 sec
      55                        30 sec
      73                        1 min
                                  go to vi. 35x
      4                           hold
                                   end
      Use H₂O as the negative control to make sure the buffer is clean.
   3. Run 2-5 μl PCR reaction to check the DNA yield.
   4. Digest DNA fragment
      Cut DNA in 30 μl reaction:
      DNA                    2-5 μl (dependent on the yield)
      Enzyme*             0.5 μl
      10x buffer           3 μl
      Cut for about 10 h.
      * Enzyme can be selected by the dCAPs program online:
      http://helix.wustl.edu/dcaps/dcaps.html
      May get incomplete digestion.
   5. Run gel and examine the PCR results.
      

Recipes

1. Tricane solution
   400 mg Tricane powder in 97.9 ml ddH₂O and use 2.1 ml Tris (pH 9) to adjust pH to 7.
   
2. 1x RAPD buffer
   1.55 ml    150 mM MgCl₂
   1.5 ml      1 M Tris (pH 8.3)
   7.5 ml      1 M KCl
   1.5 ml      0.1% Gelatine (heat gelatin to dissolve completely)
   12.05 ml
   Add 88 ml H₂O and autoclave at 121 °C for 20 min. Store at 4 °C.
3. RAPD⁺ (100 ml)
   30 μl        dATP
   30 μl       dCTP
   30 μl       dGTP
   30 μl       dTTP (each 100 mM)
   150 μl     BSA (20 mg/ml)
   Aliquot and store at -80 °C.
   
4. 2x RAPD⁺ might work better for PCR than 1x RAPD⁺
   100 ml
   3.1 ml           150 mM MgCl₂
   3 ml              1 M Tris (pH 8.3)
   15 ml            1 M KCl
   3 ml              0.1% Gelatine
   Add 75.9 ml H₂O and autoclave at 121 °C for 20 min and add 2x nucleotides.
5. PCR lysis buffer
   1x RAPD buffer
    0.3 %            Tween 20
   0.3%              NP40
   100 μg/ml      Proteinase K
   Store at -20 °C.
   

Acknowledgments

This protocol was developed in the Michael Granato Lab at University of Pennsylvania, Philadelphia, USA, and this work was supported by NIH grant R01HD037975.