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    STE Buffer Protocol

    Uploaded by

    JasminSutkovic
    This document outlines the reagents and procedure for extracting DNA from fish fins. The procedure involves homogenizing fin tissue, lysing the cells using an extraction buffer, purifying the DNA through multiple steps of phenol-chloroform extraction and ethanol precipitation, resuspending the purified DNA in TE buffer or water, treating it with RNAase, and assessing concentration and quality before using in PCR or storing at -20C. Key reagents include extraction buffer, TE buffer, phenol-chloroform-isoamyl alcohol, chloroform-isoamyl alcohol, sodium acetate, and ethanol.

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    © All Rights Reserved
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    STE Buffer Protocol

    Uploaded by

    JasminSutkovic
    297 views3 pages
    This document outlines the reagents and procedure for extracting DNA from fish fins. The procedure involves homogenizing fin tissue, lysing the cells using an extraction buffer, purifying the DNA through multiple steps of phenol-chloroform extraction and ethanol precipitation, resuspending the purified DNA in TE buffer or water, treating it with RNAase, and assessing concentration and quality before using in PCR or storing at -20C. Key reagents include extraction buffer, TE buffer, phenol-chloroform-isoamyl alcohol, chloroform-isoamyl alcohol, sodium acetate, and ethanol.

    Original Description:

    STE buffer protocol, for Fish DNA isolation

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    This document outlines the reagents and procedure for extracting DNA from fish fins. The procedure involves homogenizing fin tissue, lysing the cells using an extraction buffer, purifying the DNA through multiple steps of phenol-chloroform extraction and ethanol precipitation, resuspending the purified DNA in TE buffer or water, treating it with RNAase, and assessing concentration and quality before using in PCR or storing at -20C. Key reagents include extraction buffer, TE buffer, phenol-chloroform-isoamyl alcohol, chloroform-isoamyl alcohol, sodium acetate, and ethanol.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    297 views3 pages

    STE Buffer Protocol

    Uploaded by

    JasminSutkovic
    This document outlines the reagents and procedure for extracting DNA from fish fins. The procedure involves homogenizing fin tissue, lysing the cells using an extraction buffer, purifying the DNA through multiple steps of phenol-chloroform extraction and ethanol precipitation, resuspending the purified DNA in TE buffer or water, treating it with RNAase, and assessing concentration and quality before using in PCR or storing at -20C. Key reagents include extraction buffer, TE buffer, phenol-chloroform-isoamyl alcohol, chloroform-isoamyl alcohol, sodium acetate, and ethanol.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
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    The passage describes the process and steps to extract DNA from fish fin tissue samples.

    The main steps involve homogenizing the fin tissue, lysing the cells using extraction buffer, purifying and precipitating the DNA using phenol-chloroform extraction and ethanol precipitation.

    Incubating the sample at 37°C and 55°C helps in lysing the cells and releasing the genomic DNA. The higher temperature helps in better lysis.

    Reagents:

    http://vlab.amrita.edu/?sub=3&brch=77&sim=218&cnt=2

    Extraction Buffer 200ML

    1M Tris Cl (pH 8.0) - 2.5ml.

    0.5M EDTA (pH 8.0) - 50ml.

    Pancreatic RNAase - 5mg.

    Proteniase K - (Required).

    TE Buffer:

    Tris base - 10mM.

    EDTA - 1mM.

    Phenol: Chloroform: Isoamylalcohol (50ml)- (25:24:1).

    Chloroform: Isoamylalcohol (50Ml) - (48:2).

    70%, 100% Ethanol (Ice cold).

    Sodium Acetate 3M.

    Procedure:-

    1. Pluck out 20-100 mg of fish fin using a sterile scalpel and a forceps.

    2. Homogenize the fins in 600l of extraction buffer using sterile mortar and pestle.

    3. Collect the paste into 1.5ml Micro Centrifuge Tube(MCT).


    4. Incubate at 37C for 1 hr in a water bath.

    5. Again incubate at 55C for 1 hour in the water bath.

    6. Centrifuge at 5000 rpm for 10 minutes.

    7. Remove the supernatant put it in a new MCT and add equal volume of Phenol:
    Chloroform: Isoamylalcohol (25:24:1). Mix slowly and thoroughly by repeated inversion
    of the MCT.

    8. Centrifuge at 12000 rpm for 10 minutes and collect the top aqueous layer in a fresh MCT.
    Do not disturb the intermediate layer.

    9. Add Chloroform: Isoamylalcohol in the ratio 24:1. Mix slowly and thoroughly by
    inversion of the tube.

    10. Centrifuge the tube at 12000 rpm for 10 minutes.

    11. Collect the aqueous layer into a fresh MCT and add 0.1 volume of 3M sodium acetate
    and equal volume of ice cold ethanol (100%). Mix the solution thoroughly until the DNA
    pellet is obtained. (Now you can see the DNA clumps in the tube.)

    12. Incubate the MCT at -20C for 1 hour.

    13. Take out the tube and centrifuge at 1000 rpm for 10 minutes and decant the supernatant
    (Ethanol).

    14. Wash the tube with 70% ethanol, by centrifuging at 1000 rpm for 10 minutes and decant
    the ethanol carefully without losing the pellet. (If you find difficulty use pipette for
    removing.)

    15. Air dry the DNA pellets at room temperature by keeping the tube opened and also blot
    using filter paper / blotting paper.

    16. Resuspend the pellet in 50-100l of TE buffer or sterile triple distilled water.

    17. Add 2.0l of RNase to 100l of DNA solution and then keep the sample at 37C for 2
    hours. (For purification.)

    18. Electrophorese the sample on a 0.7% of agarose gel.

    19. Estimate the DNA concentration at 260nm. (OD at 260nm corresponding to 50g/ml of
    double standard DNA.) Concentration of sample=Dilution factor X Absorbance.

    20. Dilute the DNA to make the concentration between 10-100 g/ml.
    21. Use 1l of DNA sample for PCR(Polymerase Chain Reaction).

    22. Keep the sample at -20C or lyophilize the sample using liquid nitrogen.

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