Zebrafish is increasingly used a genetic model organism in biomedical studies. This protocol provides a detailed procedure about the identification of the genotype of an adult zebrafish or a zebrafish embryo.
Materials and Reagents
- Tricane (Sigma-Aldrich, catalog number: 886-86-2 )
- dNTPS (QIAGEN, catalog number: 201900 )
- Proteinase K (Roche Diagnostics, catalog number: 03115836001 )
- MEOH
- DNA polymerase
- MgCl2
- Tris
- KCl
- MgCl2
- Gelatine
- BSA
- NP40
- Tween 20
- PCR lysis buffer (see Recipes)
- RAPD+ (see Recipes)
- 1x RAPD buffer (see Recipes)
- 2x RAPD+ buffer (see Recipes)
- Tricane solution (see Recipes)
Equipment
- Standard tabletop centrifuges
- Incubator (55 °C and 65 °C)
- PCR thermal cycler
- 96-well PCR plate
- Razors
- Plastic plates
- Plastic beaker
- Forceps
- Spoon
Procedure
- Fin clip:
- Bring clean plastic plates, razors, Tricane, a plastic beaker, forceps and a spoon to fish room.
- Prepare a 96-well plate, with 100-200 μl of MEOH in each well.
- Add 20 ml Tricane to the beaker and add clean water to dilute the tricane (1:15 dilution).
- 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.
- Quickly put the fish back to the single box and put the tail into one well of the PCR plate with MEOH in it.
- Label both the single box and the well.
- 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.
- Genomic DNA extraction:
- Remove as much MEOH as possible.
- Incubate at 55 °C for 5 min to evaporate all MEOH.
- Add 100 μl lysis buffer per well (200 μl lysis buffer for 72 hpf embryo).
- Incubate at 55 °C O/N and heat inactivate at 95 °C for 10 min.
- Centrifuge at 4 °C at 1,000 rpm for 2-10 min.
- Store DNA at -20 °C or for long time at -80 °C.
- PCR after genomic DNA extraction
- 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). - 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. - Run 2-5 μl PCR reaction to check the DNA yield.
- 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. - Run gel and examine the PCR results.
Recipes
- Tricane solution
400 mg Tricane powder in 97.9 ml ddH2O and use 2.1 ml Tris (pH 9) to adjust pH to 7.
- 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. - 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.
- 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. - 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.
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Jing, L. (2012). Genotyping for Single Zebrafish (Fin Clip) or Zebrafish Embryo.
Bio-protocol Bio101: e182. DOI:
10.21769/BioProtoc.182.