I tried some protocols without liq. N ( because we haven't it). They didn't produce a good quality DNA yield for PCR studies.
If you have a 'freeze dryer' machine, your can freeze dry your samples first and then do DNA isolation.
I also attached this paper for you. I hope it is useful for your research project. In the paper, it described the use of a quick method to isolate rice genomic DNA without using liquid Nitrogen.
There are the Sand method for DNA extraction where you can use Sand instead of liquid Nitrogen
You can use CTAB method. It allows high recovery of DNA with excelent quality.
Dear all,
Thank you very much for your updates.
Dear Yuan-Yeu Yau, Thank you very much for your attachment. I'll try it.
Dear Ahmad, Sand method is a new one to me. If you have further details could you please put them.
Regards!
you can modified CTAB method and freezing mortar. you will get good quality and quantity from that. *from my experience
Hi Darshani,
The sand (without using liquid N2) method mentioned by Ahmed is also new to me, so I checked out 1 paper. Since you requested to see the detail of it (your previous post), this is the website you can take a look:
http://www.ncbi.nlm.nih.gov/pubmed/20957085
"Int J Mol Sci. 2010 Sep 8;11(9):3149-57.
A simple method for DNA extraction from mature date palm leaves: impact of sand grinding and composition of lysis buffer.
Arif IA1, Bakir MA, Khan HA, Ahamed A, Al Farhan AH, Al Homaidan AA, Al Sadoon M, Bahkali AH, Shobrak M.
Dear Dr Darshani Wijayawardhana,
I hope that you could have a look in my Ph D thesis it has a DNA marker part an include the DNA extraction and PCR in the following web page.
http://sundoc.bibliothek.uni-halle.de/diss-online/04/04H184/prom.pdf
the methods is as following using seeds
2.3.3.2. Genomic DNA extraction for mapping
Total genomic DNA was isolated according to the protocol described previously by
Anderson et al. (1992). Briefly, 3-5 g of leaf tissue per sample (each sample was collected from each F2 seedling 8 weeks after sowing, 81 plants in total) were ground in liquid nitrogen and incubated at 60 °C for 45 min with 15 ml of extraction buffer, (100 mM Tris-HCl, 500 M NaCl, 50 mM EDTA, 1.25 % SDS) in 50 ml polypropylene tubes. After cell disruption and incubation with hot isolation buffer, proteins were removed by chloroform: iso-amyl alcohol (24:1, v:v). Samples were incubated for 30 min by shaking and then centrifuged at 3000 rpm for 30 min. The aqueous layer was transferred to a new tube and 20 μl RNAse A (10 mg/ml) was added. Samples were incubated for 30 min at room temperature. One volume of cold ethanol was added to precipitate DNA. After 30 min incubation at 4 °C, precipitated DNA was hooked out and placed in a 2 ml reaction tube containing 1 ml of 75% ethanol. After washing twice with 75% ethanol, the washing solution was removed and the DNA pellet was dried thoroughly and dissolved in TE buffer. The DNA samples were diluted and stored at -20 °C. The DNA was diluted to a concentration of 50 ng/μl before used in SSR experiment.
I hope this method will help you
with best regards
Khaled
Thanks every one. Dear Dr. Khaled, I like to try it!
Dear Yuan-Yue Yau, thank you very much for your kind help. I'll try it for mature leaves.
With kind regards
Darshani
You get it good quality of DNA without using liquid nitrogen .
you add extraction buffer in mortar and then proper grind it and follow the simple procedure for DNA isolation
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