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This protocol explains how to extract DNA from a single zebrafish embryo.

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[Bio101] Zebrafish Embryo DNA Preparation
[Bio101] 斑马鱼胚胎DNA的制备

分子生物学 > DNA > DNA 提取
作者: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
2/20/2012, 10231 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.184

[Abstract] This protocol explains how to extract DNA from a single zebrafish embryo.

[Abstract] 该方法说明了如何从一个单个的斑马鱼胚胎中提取DNA。

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.

材料和试剂

 

1.        蛋白酶K(罗氏03115836001

2.        1MTrispH8.3

3.        氯化钾

4.        吐温2010%,EMD4 biosciences655207

5.        NP4010%,Merck492018

 

设备

 

1.        PCR

2.        离心机

3.        孵化器

 

步骤

 

1.        冷冻单活胚胎

1)      E3洗涤dechorionated胚胎3X

2)      将一个单个的胚胎放入一个96孔板,并移除所有多余的缓冲液。

(如果需要,干燥保存于-20°C)。

2.        DNA的制备:

1)      添加50μl裂解液到单个的胚胎(活体或在situ'd)。

2)      98 °C孵育10分钟裂解细胞。自旋向下。

3)      加入5μl蛋白酶K10 mg / ml的储存液)到单个的胚胎。

4)      55 °C孵育至少2个小时(长与孵育期,清除DNA

5)      98 °C孵育至热灭活蛋白酶K

6)      彻底和自旋出现碎片。

7)      单个胚胎的DNA 每次PCR rxn使用2μl

 

配方

 

1.        1X PCR缓冲液:

配制50 ml

10 mMTris - HCl pH8.3500μl 1MTrispH8.3

50 mM氯化钾(2.5 ml1 M氯化钾)

无菌水47 ml

(可在室温下保存几个月)

2.        胚胎细胞裂解液:(含有吐温20NP401X PCR缓冲液)

配制10 ml裂解缓冲液中:

1X  PCR缓冲液9.4 ml

300μlNP4010%的储存液)***现用

300μl  tween2010%的储存液),现配***

3.        E3

60X E3的股票(2L

34.4氯化钠

1.52氯化钾

5.8CaCl2.2H2O

9.8MgSO4.7H2O

加蒸馏水至2000 ml

储存在室温下。

饲养斑马鱼时稀释成1X,使用160 ml的储存液,并填加ddH2O10L

 

 

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How to cite this protocol: Jing, L. (2012). Zebrafish Embryo DNA Preparation. Bio-protocol Bio101: e184. DOI: 10.21769/BioProtoc.184; Full Text



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