I have to extract DNA from a large number of garlic leaf samples for SSR analysis. I am doing a bead milling technique using SPEX SamplePrep GenoGrinder. My current protocol involves using lyophilized garlic leaf tissue (around 50 mg), two 3.2 mm metal beads, 2 ml tubes, 1200 rpm for 45 sec with pause every 5 sec to rest tubes in ice. My extraction buffer is composed of 3% CTAB, 100 mM Tris-HCl ph 8.0, 20 mM EDTA, 4% PVP, 3% B-me, and 2 M NaCl. My problem is that all of the DNA that I get from my method is sheared/degraded. Is there anyone who has an experience using the bead milling technique for DNA extraction of fragile leaf tissue?
Thank you in advance.
Hi Grace,
Please contact Dr. Meryem Ipek, she is also a ResearchGate member (see below). We used to work together. She has done a bunch of garlic studies (see her RG profile), including plenty of garlic molecular marker (such SSR, AFLP,....) studies. She should have a good protocol for garlic DNA isolation and suitable for using in molecular marker analysis. Good luck on your research!
https://www.researchgate.net/profile/Meryem_Ipek/contributions
@ Grace,
Yes. I don't know how frequently she uses RG or response to RG email. So, if you contact her through RG email and don't get a response. You should try to fine her academic email on her publications in her RG profile. Good luck.
Hello
After CTAB adding, 25 mg of charcoal per gram of initial tissue sample was added.
and then tubes were incubated at 65 °C for at least 3h please.
Good luck
Hi Grace,
I hope you have your protocol sorted out. SPEX manufacture Aluminium blocks that you can put on ice these will keep your samples cooler longer and may help preserve your DNA.
Hello
PLease have a look this paper
Article Assessment of Genetic Diversity and Structure of Large Garli...
Best wishes
