I am extracting DNA for bulking and sequencing. A260/280 of samples range from 1.90 - 2.05, and the values are higher than A260/230. After RNAse treatment, A260/280 ranged from 1.85 - 1.96. However, when I try to check the quality of the DNA in 1% agarose, the samples appears to be degraded.
I am using CTAB method using Chloroform for extraction.
Usually A260/280 value is about 1.8-2.0 while A260/230 ranges from 2.0-2.3.
Do you mean your A260/280 value is higher than A260/280.? This quite uncommon
Also what was your sample concentration when you ran the gel? If the concentration was low, it might give you false impression that the faded band was degraded DNA.
Many DNA purification kits are available in nowadays. It make your DNA more purified from proteins and other contaminants during the processing. I also getting some errors when measuring A260-A280 values without purification.
Zhi Xiong Chong Yes, samples have higher A260/280 value than A260/230. Concentration of samples after RNAse treatment range from 250 - 1800 ng/ul.
Shan Lakmal Edirisinghe Thank you. I will try to look for DNA purification kits, although I am working with around 250 samples (for bulked DNA), is there a cheaper way to purify without using purification kits?
Hello Jonathan,
You may try to add an additional ethanol (70%) washing step to wash the pellet. This will greatly improve the quality of your DNA isolation.
Hope this helps,
Charith
Jonathan Concepcion I will suggest you change or used new elution for your samples. Maybe your elution has a problem that directly affects A260/280 or A260/230. When selecting your elution buffer, it is important to consider the requirements of your desired downstream processes. I think you're using TE.
1. DNA is not the only molecule that can absorb UV light at 260nm, RNA also has a great absorbance at 260nm, and the aromatic amino acids present in protein absorb at 280nm, both contaminants, if present in the DNA solution, will contribute to the total measurement at 260nm. Please make sure good handling while column transfer stages. Strong absorbance around 230nm can indicate that organic compounds or salts are present in the purified DNA.
2. Add additional washing step like 70% EtOH or 1x in 76% EtOH/10 mM AcNH4; then a second wash 1x70% EtOH
Hope your success through
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