Apart from conventional DNA isolation protocols and commercial kits;What sought of modifications should leads to the isolation of high quality DNA for downstream process?
That depends a lot on the type of sample as well. I have worked with tree leaves stored in minus twenty for more than a year. Grinding in liquid nitrogen and using CTAB method should be okay. Using more mercapto- and proteinase-K can also help. Increasing NaCL buffer concentration usually helps slimy (polysaccharides?) leaf extracts. In the end, you would need to try couple of existing protocols and modify to suite to your needs.
CTAB method have been tried before by my lab mates... still got DNA although degraded. Degraded DNA maybe due to improper storage practice
Yes Asmuni you're right. CTAB does well with many samples. Long term storage might impact quality of DNA. That depends on the quality we need. For advance sequencing etc. degraded DNA should be a big problem. RAPDs and ISSRs (or even AFLPs) work well with a lesser quality DNA, but not for sequencing. A final DNA cleaning with salt precipitation sometimes works if we need to use stored samples. Still recommend liquid nitrogen and Proteinase-K.
Yes as mentioned by all friends either CTAB or Dellaporta also work well.
First of all really not followed the sentence 'leaf samples stored in deep freezer(-20 degree) for long time'. As per my knowledge we try to store samples carefully years or months in -70C. No question to feel if you take enough care while keeping/storing the leaves in the sense once they are plucked from the plant.
Fresh samples always are the best. The more you store the more DNA gets degraded. But, if it is possible to store in liquid nitrogen, that has no parallel. Still, if the stored ones are all we have, Proteinase K treatment, high molar NaCL precipitation, repeated Isoamyl-alcohol extraction, Ribonulease incubation and finally, a salt precipitation by any commercial kit should be the best way I feel to get a about 1.8 reading in 260/280 and an un-smeared gel. But, things change a lot with time, more protocols may be available. What I mentioned is inexpensive and easy to handle for a large-scale extraction work. All the best.
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