When i check RNA purity the peak height at 260 or 280nm is less than the peak height at 230nm or other nm.
Hi Govardhan,
phenol, TRIzol, chaotropic salts, and other aromatic compounds absorb between 200nm and 230nm. Some downstream procedures may work perfectly while others may give problems. It could happens when you harvested the aqueous phase. You need extra step of purification (re-precipitation, re-washing and resuspending). the optimal rang of ratio are:
DNA 260/230: 1.7-2;
260/280: 1.7-2 (it is less than RNA ratio because is more probably that to have some DNA still bounded to Histone proteins.
RNA 260/230: around 2;
260/280: around 2.2 (high purity to avoid the presence of RNAse that could to degrade your sample).
Hi Shaifali, you have problem with DNA extraction from Mycobacterium tuberculosis because its wall is rich of micolic acids and other compound and the yeld could be poor. You need to use lysozyme (20 mg/ml and incubated at 37°C for 2 hours) before DNA extraction.
260/280 value should be somewhere in a range of 1.9-2.4, ideally at 2.2. If so, your DNA purity is good.
Pure DNA must have a 260/280 ratio between 1.8 and 2. DNA is detected at 260 and protein at 280 nm.
Protein absorbs at 280 while nucleic acids absorb at 260. The 260/280 ratio is an indicator of purity from protein contaminants. As a general rule of thumb, DNA should have a 260/280 ratio approaching 1.8, while RNA should have a 260/280 ratio nearer to 2.0-2.1. The 260/230 ratio is another indicator of sample purity and should be around 2.0-2.2, and in all instances should be greater than 1. The high peaks you're seeing at 230 are indicative of a contaminant, such as phenol or chaotropic salts. I would advise you to re-precipitate your RNA to try to remove the contaminants. Good luck with your research!
I am also having the same problem with extracted DNA of Mycobacterium tuberculosis. But I am having range of 1.2- 1.4 and also in 260/230 reading there is a range of 1.03 to 1.22. So what does it means?
Hi Govardhan,
phenol, TRIzol, chaotropic salts, and other aromatic compounds absorb between 200nm and 230nm. Some downstream procedures may work perfectly while others may give problems. It could happens when you harvested the aqueous phase. You need extra step of purification (re-precipitation, re-washing and resuspending). the optimal rang of ratio are:
DNA 260/230: 1.7-2;
260/280: 1.7-2 (it is less than RNA ratio because is more probably that to have some DNA still bounded to Histone proteins.
RNA 260/230: around 2;
260/280: around 2.2 (high purity to avoid the presence of RNAse that could to degrade your sample).
Hi Shaifali, you have problem with DNA extraction from Mycobacterium tuberculosis because its wall is rich of micolic acids and other compound and the yeld could be poor. You need to use lysozyme (20 mg/ml and incubated at 37°C for 2 hours) before DNA extraction.
Hey Govardhan,
Check this out:
http://www.nanodrop.com/Library/T009-NanoDrop%201000-&-NanoDrop%208000-Nucleic-Acid-Purity-Ratios.pdf
I agree the statement written by Juanita....Same problem, protein contamination in my RNA samples. I tried purification by ethanol re-precipitation and washing. But RNA sample concentration got reduced around 50%. Need a better protocol.
Is it Phenol:chloroform:isoamyl 25:24:1 step will help to remove proteins and chaotropic salts?
If you are using the Phenol:chloroform:isoamyl 25:24:1 extraction technique. When you are removing the aqueous phase even if you are very careful you can still draw up very small amounts of the interface without noticing. Increasing the scale of your extraction and only taking half of the aqueous phase can help avoid this. Not to mention optimizing your extraction cell density to prevent excess amounts of DNA, protein and other cell debris which will increase the chance of protein contamination.
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