The genomic DNA,I isolated from turmeric using ctab .The protocol followed for genomicDNA isolation is: grind turmeric leaves using DNA extraction buffer(100 mM tris PH 8,20 mM EDTA,1.4 M Nacl,2% CTAB)and PVP without using liquid nitrogen,then incubate at 65 degree celsius for 1 hour,followed by two times extraction with chloroform and isoamyl alcohol and addition of isopropanol with overnight incubation.This is followed by centrifugation,ethanol wash of pellet.The dried pellet is finally dissolved in nuclease free water.The absorbance value AD260/280 of DNA is around 2-2.3.The PCR done using this DNA is not giving bands in most cases using ssr primers.I doubt it's due to DNA quality.Can someone help me out with the DNA isolation protocol for turmeric?
Hi Aswathi ,
There are some reasons we may not get PCR products:
- Not good primers
- Not good PCR reactions
- Not good DNA (quantity and quality)
You have to make sure you have good primers and PCR reactions by testing with control DNA. After confirming those parameters you should check your DNA.
Normally, you can do gel running to check your DNA.
+ If they have strong and sharp bands on the gel, you can measure DNA concentrations and dilute to 50-100 nanograms/microliters and use 2 microliters for PCR reactions.
+ If they do not have sharp band or they have only smear bands, you need to re-isolate your DNA.
Your protocol looks OK, but I recommend you should use liquid nitrogen for grinding your samples.
Good luck,
Phat
Hi,
Thanks a lot for the answer.I have a doubt since I'm using manual method for dna extraction,does grinding a lot cause shearing of dna?
Hi Aswathi,
If you grind your samples in liquid nitrogen, you do not have to care about grinding time. Fine powder of samples in liquid nitrogen should result good DNA. However, if you do not use liquid nitrogen, longer grinding time should be a problem for your DNA extraction.
Best
Phat
We incubate overnight with isopropanol to precipitate DNA after extraction with chloroform and isoamyl alcohol.
hi Aswathi A.P
this protocl that i mentioned you is one the best dna extraction method its perfect i hope it will be useful for you
Hi Reyhane,
First of all,Thanks a lot for sharing the protocol.I would like to know how much concentration you get for DNA using this protocol?
The observation that the AD260/280 absorbance ratio of your samples is around 2-2.3 indicates a heavy contamination with RNA. While usually RNA is generally not a problem for PCR in some cases it may still interfere with the enzymatic procedures. Maybe that after the precipitation step you treat the samples with RNAse-A (15 min), then add Protease for 2 hours. The samples should then be deproteinated with a phenol-chloroform-isoamylalcohol extraction.
Best wishes
Ales
Hello Aswathi A.P ... I have isolated DNA using modified CTAB protocol from many wild and cultivated Zingiberaceae including turmeric (multiple accessions, I have published a paper on that). It is a simple protocol with the total time of extraction being about 4 hours (no need for overnight incubation). All the isolated DNA samples were stable for 4 years. I did RAPD, ISSR and gene specific marker sequencing (matK, rbcL, ITS) from the DNA.
If you want, you can find it in my profile ( Article A fast and reliable method for DNA extraction from different...
)For any further query, please feel free to contact me.
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