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Zebrafish Embryo DNA Preparation   

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Abstract

This protocol explains how to extract DNA from a single zebrafish embryo. It does not require the use of expensive kits.

Materials and Reagents

  1. Proteinase K (Roche Diagnostics, catalog number: 03115836001 )
  2. 1 M Tris (pH 8.3)
  3. NaCl
  4. KCl
  5. CaCl2·2H2O
  6. MgSO4·7H2O
  7. Sterile water
  8. 10%Tween 20 (EMD Biosciences, catalog number: 655207 )
  9. 10% NP40 (Merck KGaA, catalog number: 492018 )
  10. Embryo lysis buffer (see Recipes)
  11. 1x PCR buffer (see Recipes)
  12. E3 (see Recipes)

Equipment

  1. PCR Thermal cycler
  2. Centrifuges
  3. Incubator
  4. 96-well plate

Procedure

  1. Freezing single live embryos
    1. Wash dechorionated embryos 3x with E3.
    2. Place a single embryo into a well of a 96-well plate and remove all excess buffer.
      (Store dry at -20 °C if needed).

  2. DNA preparation:
  1. Add 50 μl lysis buffer to single (live or in situ'd) embryos.
  2. Incubate at 98 °C for 10 min to lyse cells. Spin down.
  3. Add 5 μl Proteinase K (10 mg/ml stock) to single embryos.
  4. Incubate at 55 °C for at least 2 h (longer the incubation, cleaner the DNA).
  5. Incubate at 98 °C to heat kill Proteinase K.
  6. Vortex thoroughly and spin down debris.
  7. Use 2 μl of single embryo DNA per PCR reaction.

Recipes

  1. 1x PCR buffer
    For 50 ml
    10 mM Tris-HCl (500 μl of 1 M Tris) (pH 8.3)
    50 mM KCl (2.5 ml of 1 M KCl)
    47 ml sterile water
    (Can be stored at RT for severalmonths)
  2. Embryo lysis buffer (1x PCR buffer with tween 20 and NP40)
    For 10 ml of lysis buffer
    9.4 ml 1x PCR buffer
    300 μl NP40 (10% stock) ***Make fresh
    300 μl tween 20 (10% stock) each time***
  3. E3
    60x E3 stock (2 L)
    NaCl                 34.4 g  
    KCl                   1.52 g
    CaCl2·2H2O      5.8 g
    MgSO4·7H2O    9.8 g
    Add distilled water up to 2,000 ml.
    Store at RT.
    To dilute to 1x for rearing zebrafish, use 160 ml of stock and fill to 10 L with ddH2O

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.

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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Jing, L. (2012). Zebrafish Embryo DNA Preparation. Bio-101: e184. DOI: 10.21769/BioProtoc.184.
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Q&A
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If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

Özge Çark
Izmir Biomedicine of Genome Center
Hi,

I tried your protocol to extract gDNA from a single embryo for mutation screening. Although my primers are specific for the target region at 60C annealing temp in my other PCRs, when I use gDNA from this protocol, I obtained nonspecific bands. What could be happened in the reaction? Can I fix this problem by adjusting annealing temperature?

Best regards,

Özge
7/12/2019 4:27:10 AM Reply
ajay verma
centre for cellular and molecular biology

Hi,
This happened with one of my primer pair too but worked by adjusting the annealing temperature. It might be due to high salts.
Good luck,
Ajay

7/16/2019 12:39:38 AM Reply


Özge Çark
Izmir Biomedicine of Genome Center

Thank you :) I will try to adjust annealing temp.

7/16/2019 4:22:42 AM Reply


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