Taking 5 ml blood i should get a good yield of DNA as the birds have nucleated rbc can anyone please tell me a proper phenol choloroform method to solve it
If you do extration form nucleated rbc only phenol method is not enough. You need to destroy RBC membrane. As you get hemolisate you can perform chloroform method.
Wash the reed blood cells with pbs buffer to remove plasma protein. Then add destiled water to destroy reed blood cell membrane or you can use ultrasonic, or another lysate
Then:
For the DNA extraction, 1.0 mL samples was transferred into eppendorf tube and centrifuged at 13,000 g for 10 min at 4 °C. The supernatant was discarded and the pellet was resuspended in 700 μL of TE Buffer (10 mM Tris-HCl, 1mM ethylenediaminetetraacetic acid; pH 8.0). Then, 50 μL of 20% sodium dodecyl sulfate (SDS) was added and incubated for 1 h at 55 °C. Following the incubation, 500 μL of chloroform-phenol was added and samples were incubated for 5 min at 45 °C followed by a cool down for 5 min at room temperature. Samples were then centrifuged at 13,000 g for 10 min at 4 °C, and the upper aqueous (DNA) supernatant was transferred into a new tube. Then, 95% ethanol (2 volumes relative to the sample volume) was added and mixed by inversion, following by incubation for 10 min on ice and centrifuged at 13,000 g for 10 min at 4 °C. The excess of ethanol was carefully removed to avoid disintegration of pellet. Samples were centrifuged at 13,000 g for 5 minutes and the supernatant was removed and allowed to air-dry for 15 minutes. Finally, The DNA pellets were suspended in TE bufferr and stored at -20 until further use.
- Badges
- Science method