Hi everyone
recently I have been isolating DNA from tobacco leaves for southern blot using modified C-TAB method. I get good quantity of the DNA but the quality is poor. Especially the 260/280 ratios aren't good and this contamination is inhibiting downstream restriction digestion as well. The current the method I am using, uses C-TAB/Cholo:IAA treatment thrice(2% C-TAB once and 10%C-TAB twice) which makes it very time and material consuming process. Does anyone knows any method especially designed for tobacco DNA isolation? your suggestions are most appreciated.
To my knowledge, CTAB is a good method for extracting the DNA from tobacco and other crops as well such as suger cane, tomato, petunia and others. Try to focus on some steps especially when adding chloroform where you are separating the DNA from plant debris. Be sure not take some debris while pulling the DNA.
Now, there are some kits available like DNAmaxi plant extraction kit where you can get pure DNA easily.
Article A Simple and Rapid Method For DNA Extraction From Leaves of ...
https://wiki.london.hackspace.org.uk/w/images/8/8d/Simple-Chelex-plant.pdf
Thank you Adel. Ali as you said my plant extract is seems to be the problem as I do not get clear aqueous phase even after three chloroform treatments. Could you please suggest anything about organic extraction? Right now I am using 200mM tris pH8. But we use 4500g for centrifugation ( unfortunately that's the maximum we have got for 50ml tubes!) does that makes any difference? How much amount of starting material should be enough? Does the age of the leave matters? I.e. If I use young leaves will that gives more phenolics? Currently I am using only chloroform:IAA for phase separation. Using phenol:chloroform:IAA will be more useful? Sorry for too many questions!
Thank you Ali for your insightful information. I will try out the possibilities and will be back with what works best. Have a lovely day.
you can use conventional CTABE for isolation from tobacco leaves its shown good result as compare to other methods
Hello everyone,
After many combinations, what worked for me is, first removing the debris by using Tris-EDTA buffer, then 2% C-TAB with 1% ME @65 degree for 1 hour, then 10% C-TAB with 0.7mM NaCl, phenol:chloro:IAA, then only Chloro:IAA. It is still very time consuming, but in the end I got moderate yield with relatively pure DNA. Using SDS instead of C-TAB was equally effective but the yield was low. spooling was not that effective had to do ethanol precipitation. Using young leaves considerably reduces pigments present in aqueous extract.
I hope that helps.
Thank you Ali once again.
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