Hi everyone,
I am working in cowpea genetic diversity and I have an issue with DNA extraction, I always get a bad quality of DNA with a lot of contamination.I am looking for a best protocol to purify it.
Thank you for your help,
First of all, treat the DNA with RNase enzyme for ~1 hr then with Proteinase K enzyme for another ~1 hr (both at 37 Celsius)
Follow the general process of DNA extraction (from step of Phenol: chloroform method).
I think you will get good quality of DNA
If your extracted DNA is contaminated with protein, you can follow the following protocol -
1. Take 100 ul of stock DNA in 2 ml centrifuge tube
2. Add equal volume of phenol:chloroform:isoamyl alcohol (25:24:1)
3. Thoroughly mix by inversion
4. Spin at 8000 rpm for 15 minutes at 10oC
5. Transfer the supernatant in a new tube
6. Add 200 ul of pre-chilled isopropanol/ethanol and 50 ul of 5M NaCl
7. Incubate at -20oC for at least 1 hr
8. Spin at 8000 rpm for 15 minutes at 10oC
9. Discard the supernatant
10. Wash the pellet twice with 76% ethanol
11. Air-dry the pallet at 37oC until the ethanol smell goes-off
12. Resuspend the pellet in TE buffer
When you went to extract DNA from plant l prever to extrasct it from the leaf
Extract it manual using CTAB ....protocol
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