Hi, I have been trying to extract Plant genomic DNA via CTAB method and I am getting a 260/280 ratio ranging between 1.3 to 1.6. Any idea how to modify the protocol for fabaceae family plant members?
My protocol involved CTAB+beta-mercaptoethanol+PVP followed by extraction via PCI, isoamylalcohol and ethanol. Dissolving the pellet in the TE buffer.
I need the DNA for sequencing.
Thanks in advance.
You should repeat chloroform:isoamyl alcohol (24∶1, v/v) steps until you see complete separation of layers.
@ozgur altundas I saw complete separation of layers in the first step itself that's why didn't repeat.
Sakshi Shekhar Mehta is the concentration of extracted DNA sufficient for your further analysis? If the concentration is okay, you may decrease the amount of the plant sample you use for extraction. This way, you will be challenged by relatively fewer possible contaminants such as protein and phenol that might cause the lower 260/280 ratio during DNA extraction.
Özgür Altundaş Hi, after isolation of my DNA I ran a gel (0.8%) and I saw the DNA inside the well, and it did not migrate even after 30 mins. What could be the reason? Contamination?
Hi Sakshi Shekhar Mehta It is not about contamination. Just because it is gDNA, means that it has a high molecular weight. Thus, the migration is slow. What is the voltage? What is the distance between electrodes as cm? I have shared one of my gDNA gel electrophoresis results below.
Özgür Altundaş I ran gel twice, one on 100 V for 45 mins the DNA remained in the wells. Second time I ran it on 80 V for 1.5 hours I could see faint bands out of the well. Your gel picture is awesome. Can you share your DNA isolation protocol if possible? and which tissue (and how many days old?) do you use?
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