I have extracted DNA from Cacao leaf tissue using CTAB, chloroform isoamyl alcohol method. When I quantified my samples on Nandodrop they show a conc. of 300-800 (ng/ul) in 50 ul of TE buffer. The ratios of 260/280 are good and 260/230 are fair.
However, when I ran my samples on 1% agarose gel electrophoresis at 70 V, I do not see any bands of DNA but only RNA. I have attached the picture of both gel and nanodrop results.
What must have gone wrong? Can you please advice me on what to do?
Regards
Gurpreet
Hi,
it could either be RNA or your DNA gets fragmented during the processing. I would propose to use dedicated kits are plant materials can give issues during isolation due to the enzymes in the vacuoles. In addition depending on your downstream workflows you should keep in mind that an inhibitor resistant chemistry might be beneificial. Let me know if you need more details on this
Sven
Hi Gurpreet,
I wonder if your samples are not contaminated with aromatics (as phenol) that mimic DNA when trying to measure the DNA concentration.
What I did not understand is what was your extraction procedure. did you want to extract separately DNA and RNA as it is currently done? or do it by the same way with the same protocol at once?
I saw you used the NanoDrop which is a good machine, very good but old since the new one can detect aromatics and tell you why you can't see bands on gel.
fred
Hi Fred, I Used CTAB DNA extraction protocol. The purpose was to get DNA for using it in Diversity arrays technology analysis. I was only after extracting DNA.
I am still wondering, Does this means I have no DNA in my samples?
Hi Gurpreet,
as you can see on your gel, the last 9 lines do not contain DNA, or too few to see it on gel.
fred
Dear G Singh. I also expect DNA degradation through Extraction steps. In addition you should reduce the conc. of agarose to 0.8 or little less due to genome volume of plants. While you should check (pH: 8.2)of working solution.
Before PCR, you should do both quantity and quality test of DNA samples. Here you can use Any standard kit protocol.
Hi in addition I wonder if the measured concentrations for plant DNA are expected to be so high? Normally all people I talked to have rather ranges of 20-30ng/µl which would further support the theory that the absorption is due to impurities
Sven
Hi Sven,
Thanks for your answers. I guess the high concentrations might be due to impurities. However, i was expecting to see some
DNA on the gel as the graph shown by Nanodrop was quite good.
Hi Gurpreet,
Try using a standard DNA extraction kit because isolation of DNA requires precised amount of all the chemicals you are using in the process. As far as the gel is concerned I use 0.8% agarose gel for my samples. You can try that. Your nanodrop reading must be due to some contamination. Also pH, concentration, amount of chemicals, precipitation time and hot-water incubation temperature has significant role in isolation procedure. You can also increase or decrease precipitation time and incubation temperature according to your sample and observe if it helps.
Give a longer time to the gel electrophoresis
Even the marcher is not clear
Hi Gurpreet,
As per my knowledge the higher conc is due to free nucleotides..the DNA sometimes undergoes degradation due to over grinding of the samples
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