DNA isolation - (May/30/2006 )
I am isolating the DNA from Bradyrhizobium.japonicum, one nodulation bacterial of soybean by using DNeasy Kit from QIAGEN, but a large amount of lipid-like materials often interfere with spin column. Would anyone like give me suggestions?
Thanks in advance
well. For trizol RNA extraction, they tell :
If a sample is known to have a high content of proteoglycans and/or polysaccharides (such as rat liver, rat aorta, plants), the
following modification of the RNA precipitation step should remove these contaminating compounds from the isolated RNA: Add 0.25 ml of isopropanol to the aqueous phase followed by 0.25 ml of a high salt precipitation solution (0.8 M
sodium citrate and 1.2 M NaCl (no pH adjustment necessary)) per 1 ml of TRIzol used for homogenization. Mix the
resulting solution, centrifuge, and proceed with isolation as described in the protocol.
This modified precipitation effectively precipitates RNA and maintains proteoglycans and polysaccharides in a soluble form. To
isolate pure RNA from plant material containing a very high level of polysaccharides, the modified precipitation should be
combined with an additional centrifugation of the initial homogenate.
In general, it is not recommended to use the high-salt precipitation if polysaccharide or proteoglycan contamination is not a
second info :
OPTIONAL: An additional isolation step may be required for samples with high content
of proteins, fat, polysaccharides or extracellular material such as muscles, fat tissue, and
tuberous parts of plants. Following homogenization, remove insoluble material from the
homogenate by centrifugation at 12,000 ×××× g for 10 minutes at 2 to 8°C. The resulting pellet
contains extracellular membranes, polysaccharides, and high molecular weight DNA,
while the supernatant contains RNA. In samples from fat tissue, an excess of fat collects as
a top layer which should be removed. In each case, transfer the cleared homogenate
solution to a fresh tube and proceed with chloroform addition and phase separation as