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STE Buffer Protocol

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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391 views3 pages

STE Buffer Protocol

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.

Uploaded by

JasminSutkovic
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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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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