Iam finding it hard to get amplification post whitefly DNA extraction using lysis buffer method. Any other simple methodology. What are the precautions or troubleshooting measures to be followed? I use a micropestle to grind the whitefly in buffer within an eppendorf tube. the whitefly gets evenly grounded which is indicated by the cloudy appearance of the buffer. which step to troubleshoot?
I would be very glad if someone could explain in brief with all components needed rather than sending a research paper link. If there is a lucid research paper explaining the protocol do send.
For less quantities of sample, you could try ChargeSwitch DNA kit from Thermo or make your own phenol-CHCl3 extraction buffer (works best).
For phenol-based DNA extraction -make phenol pH 7-8 (to prepare this, equilibrate 10ml phenol two times with an equal vol of 1M Tris pH8, followed by once with 100mM Tris pH8). You can directly grind flies in the phenol+Tris buffer. for every 500uL of (Tris-buffer+Phenol) add 200uL ChCl3, centrifuge and wash sup once more with 250uL CHCl3. Pellet DNA with 1/10 vol 3M sodium acetate + 2vol Ethanol, Wash with 70% ethanol, centrifuge and air dry pellet for 10 min. Dissolve DNA in 20-30uL Tris EDTA pH 7.4/8 at 50deg C for 5 min.
Hope this protocol works. Make sure that the buffers have pH more that 7.4. Good Luck!
Use classical phenol-chloroform-isoamyl alcohol method. Tons of protocol available on net.
you can try, but according to my knowledge, CTAB method is mainly used for the plant material. Of course you can try and check the amount you get, because there will definitely DNA in your extraction. Phenol method is universal, you can also try that.
- Badges
- Science method