I am looking for a protocol to extract DNA from damselflies preferably inexpensive manual extraction procedure. I do not have enough money to buy expensive DNA extraction kit
Dear Kawsar
Kindly follow this link it might be of use to you
https://www.researchgate.net/post/which_method_is_good_for_insect_DNA_isolation
Hello Pranay,
Here is your protocol, we are working with it since 5 years... let m know for any further issues
DNA EXTRACTION PROTOCOL FOR INSECTS
1. Dry the alcohol dipped samples, transfer it to microcentrifuge tubes.
2. Add 60 0C pre-warmed CTAB (600ul) +3ul of Beta-Mercaptoethanol+ 10ul of 20% SDS
3. After crushing add 3ul 0f Proteinase-K in each tube.
4. Vortex each tube for 5 minutes vigorously.
5. Keep it overnight incubation, and vortex it after the interval of 2-3 hours if possible, after incubation, cool the samples to room temperature, and centrifuge at 14,000 rpm for 10 min
6. Add equal volume of Phenol: chloroform: Isoamyl alcohol (25:24:1) and mixed it for 5 minutes (vortex).
7. After mixing, centrifuged at 14,000 rpm for 10 minutes.
8. Then, take supernatant fresh microfuge tube and discard the pellet.
9. Add Chloroform: isoamyl alcohol (24:1), 600ul in each tube and mix it (vortex).
10. Centrifuge at 12,000 rpm for 10 minutes.
11. Take the supernatant in fresh microfuge tube and discard the debris.
12. Add chilled isopropanol (400 ul) and mixed slowly until white flakes appear.
10. Then keep it at -35 or-50 0C in deep freeze for 1hour.
11. Brought the sample at room temperature.
12. Centrifuge at 10,000 rpm for 10 minutes.
13. Decant the supernatant and add 70% chilled ethanol (400ul) +ammonium acetate (100 ul) to the pellet for washing.
14. Centrifuge at 10,000 rpm for 10 minutes.
15. Decant the supernatant carefully; add 400ul of absolute alcohol and Centrifuge at 10,000 rpm for 10 minutes.
16. Decant the supernatant and dry the pellet at room temperature.
15. Dissolve it in nuclease free water.
CTAB EXTRACTION BUFFER – (For 50ml)
1M Tris-HCl 5ml
0.5 M EDTA 2ml
5M NaCl 17.5ml
10% CTAB 10ml (5 gm in 50 ml)
Distilled Water 15.5 ml to make up the volume.
Set the pH to 7.5-8.0. (Autoclave the whole content before use.)
At number 13, you have used ammonium acetate, please mention the molarity of ammonium acetate. Thanks
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