Genotyping Transgenic Zebrafish Using Genomic DNA Extracted from Clutch of Embryos    

Abstract

Materials and Reagents

1. Zebrafish embryos
2. MeOH
3. Phenol
   
4. Chloroform
5. Isoamyl alcohol (IAA)
6. NaCl
7. KCl
8. MgCl₂
9. EtOH
10. TE
11. Tween 20
12. NaOAC
13. Gelatine
14. NP40
15. Proteinase K
16. ddH₂O
17. Phenol: chloroform: isoamyl alcohol (25:24:1)
18. Primers (custom ordered from IDT)
19. 1x RAPD buffer (see Recipes)
20. RAPD⁺ (see Recipes)
21. PCR lysis buffer (see Recipes)
    

Equipment

1. PCR thermal cycler
2. Incubator
3. Glass pipette
   
4. Remove MeOH and dry embryos in 55 °C incubator.

Procedure

1. Collect embryos
   Groups of 25-40, 1 transgenic embryo in clutch of 40 embryos should be detected, fix in MeOH, store at -20 °C.
   1. Remove MeOH and dry embryos in 55 °C incubator.
   2. Add 10 μl/embryo of lysis buffer (w/ Rnase & Dnase).
   3. Incubate at 37 °C, overnight.
   4. Extract with 1 volume (vol.) of phenol: chloroform: isoamyl alcohol (25:24:1); vortex 15 sec and spin for 1 min and then extract top aqueous layer.
      
   5. Repeat phenol: chloroform: isoamyl alcohol extraction.
   6. Extract with 1 vol. of chloroform: IAA (24:1).
   7. Add NaCl to 0.3 M (*Do not use NaOAc as it will precipitate DNA into a slurry!).
   8. Add 2 vol. of cold EtOH (should see DNA precipitate into cloud).
   9. Prepare glass pipette with hook at the end.
      
   10. Remove DNA by stirring glass pipette into Eppendorf.
       
   11. Carefully wash or remove pellet from pipette with 70% EtOH and into new eppendorf; dry pellet.
   12. Dissolve DNA into 100 μl of TE or ddH₂O. 
   13. Add 10 μl NaOAC and 220 μl EtOH.
       
   14. Spin for 15 min at max speed at 4 °C.
   15. Wash pellet with 70% EtOH and spin at max speed at RT for 5 min.
       
   16. Dry pellet and resuspend in ddH₂O (i.e. 50 μl).
       
       
2. Transgene PCR detection
   
   1. To detect transgene, using primers that span the promoter and cDNA sequence or primers that span the Tag sequence such as Myc, GFP if used in the transgene.
   2. PCR set up
      X μl       RAPD⁺ (make up to 20 μl)
      2 μl       DNA
      1 μl       F primer (20 μM)
      1 μl       R primer (20 μM)
      1 μl       Tag polymerase
      
   3. PCR program
      94 °C              1 min
      94 °C              30 sec
      54 °C              2 min
      73 °C              1 min
                             go to ii. 5x
      94 °C              30 sec
      55 °C              30 sec
      73°C               1 min
                             go to vi. 35x
      4 °C                hold
                             end
      Run 2-5 μl pcr to check the yield.
      

Recipes

1. 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.
   
2. 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.
   
3. 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 modified from the original protocol by Julie Lefebvre and developed in the Michael Granato Lab at University of Pennsylvania, Philadelphia, USA. This work was supported by NIH grant R01HD037975.