I want to conform T-DNA insertion cause abnormal embryo, then extracting DNA is needed. Please help me!
The following literatures should help you.
http://www.shigen.nig.ac.jp/rice/rgn/vol23/b25.html
Good Luck
you try with simple CTAB method or even Lysis buffer contains EDTA, SDS and TRIS HCL will yield good quality DNA
Dear Dr Tuyet Van,
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 from embryo
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
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