How to eliminate visous in leaves during DNA isolation - (Mar/20/2008 )
I got a problem when isolating DNA from leaves of forest trees. I am using CTAB method.
The extract solution has green color and very viscous. It happens even at last step of protocol. The DNA after dilute in TE also got the same matter.
Does anybody know to to eliminate it?.
I think that's probably high amount of polysacharides. Do you use phenol:chloroform in that protocol?
working with forest trees can be a pain...once tried to extract RNA from cambium tissue in Fagus grandifolia and in 4 months could not get any. you could use columns (if available), that should facilitate things a lot.
Do you use fresh material? How much g of tissue for each extraction?
Did you try to start from a smallest amount? Probabily this trick will help you to eliminate better all the undesired componenets...
Let us know!
I am using fresh forest leaves and also used phenol:chloroform: isoamyl alcohol (25:24:1). Unfortunately, it did not success to eliminate viscous problem.
200 mg for each sample is used and i amd using CTAB extraction protocol
Any ideas are appreciated
If it's polysaccharides, I would try to make more washes with NaCl 2.5M.
Once you performed the isopropyl alchool step:
1* Add wash buffer (ethanol 76% + ammonium acetate 10 mM), transfer everything (pellet + aqueous phase) in a clean tube.
2* Centrifuge 12000 rpm 1’, then discard liquids and dry pellet under a fume hood (or leaving the tube 5'-15' in thermomixer 50-60°C).
3* Resuspend pellet in 400 μl sterile mQ H2O then transfer to a new 2 mL tube.
4* Add 1 volume of NaCl 2,5M and centrifuge 13200 rpm, 10’.
5* Collect the upper phase and put it in a new tube.
6* Add 2/3 volume of cool isopropanol (isopropyl alcool)
7* Centrifuge 7500 rpm, 20’ (the nucleic acids will pellet on the bottom of the tube)
8* Discard liquids and repeat steps 3-7 one or two times.
After 2-3 whashes with NaCl, you can:
* repeat till step 3 (you resuspend your pellet in 400 μl sterile mQ H2O) and go on with RNAse or proteinase K steps (if desired)
* go on till step 5, add 0,6 volumes of isopropyl alcool (DNA becomes evident as a pale mass suspended in the solution), leave samples 30' at –20°C, centrifuge 13200 rpm 30’ and pellet DNA in the tube as usual.
I hope I was of some help!