Rihab,

Abnormal value (high or low) of 260/230 may indicate problem with a sample or with extraction procedure. This info may help

1. A low A 260/A230 ratio may be the result of:

• Carbohydrate carryover (often a problem with plants).

• Residual phenol from nucleic acid extraction.

• Residual guanidine (often used in column based kits).

• Glycogen used for precipitation.

2. A high A260/A230 ratio may be the result of:

• Making a Blank measurement on a dirty pedestal

• Using an inappropriate solution for the Blank measurement. The blank solution should be the same pH and of a similar ionic strength as the sample solution.

Example: Using water for the Blank measurement for samples dissolved in TE may result in low 260/230 ratios.

Good luck

Reference:

William W. Wilfinger, Karol Mackey, and Piotr Chomczynski,

Effect of pH and Ionic Strength on the Spectrophotometric

Assessment of Nucleic Acid Purity: BioTechniques 22:474-481

(March 1997)

Hi, how is the ratio? Of course, contaminants can inhibit a PCR. Cheers, Nadine

Hi,

The ratio in these samples ranged between 0.4- 0.7

Rihab,

Abnormal value (high or low) of 260/230 may indicate problem with a sample or with extraction procedure. This info may help

1. A low A 260/A230 ratio may be the result of:

• Carbohydrate carryover (often a problem with plants).

• Residual phenol from nucleic acid extraction.

• Residual guanidine (often used in column based kits).

• Glycogen used for precipitation.

2. A high A260/A230 ratio may be the result of:

• Making a Blank measurement on a dirty pedestal

• Using an inappropriate solution for the Blank measurement. The blank solution should be the same pH and of a similar ionic strength as the sample solution.

Example: Using water for the Blank measurement for samples dissolved in TE may result in low 260/230 ratios.

Good luck

Reference:

William W. Wilfinger, Karol Mackey, and Piotr Chomczynski,

Effect of pH and Ionic Strength on the Spectrophotometric

Assessment of Nucleic Acid Purity: BioTechniques 22:474-481

(March 1997)

Dear Rihab

The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm.

You may read the following article.

Best Regards

Hi,

Thank you all for the good advices and the informations you gave.

Rehab,

You may consider Qubit 2.0 Flurometer instrument (Invitrogen now Life Technologies) to quantify accurate DNA , RNA, and Protein concentration.

Good luck

The OD ratio of DNA extracts has limited to no correlation with the amplificability of your extract (Ramos-Gómez et al., 2014, Costa et al., 2010; Muzzalupo et al., 2007; publications about the extraction of DNA from vegetable oils).

In the paper of Ramos-Gómez et al. they had some ratios of as low as 0.8 and the PCR did well nontheless. It's another sample matrix, but the observation should be applicable in your case too.

Thank you all for the vote and Rehab for given us a chance to discuss on the issues that matter for the cause of science, knowledge, Research. and Development.

Samples with a low 260/230 (below about 1.8) have a significant presence of these organic contaminants that may interfere with other downstream processes like RT-PCR or the IVT in Affymetrix® experiments, lowering their efficiency

I have often low ratio of OD260/230 of my RNA samples. No matter if I isolate it from Trizol or Kits (Qiagen and Sigma). I collect mammary tissue in RNA later. Bring them to lab at RT and store at 4 deg Overnight and finally transferred to -80 deg C. We process all these with in two week of storage. Still failed to understand why my ratios are bad (

Did you tried to collect the samples in liquid nitrogen? Or process it immediately in ice cold trizol?

We flash freeze all of our fresh tissue that is for RNA studies.  The shorter the amount of time before freezing, the better quality we get; we begin to see RNA degradation even after 30 min to 1 hour of sitting at room temp.  

No,I did not tried liquid nitrogen nor ice cold Tripoli.

Rihab, a high 260/230 ratio cannot "inhibit the amplification of the DNA to form a band on the agarose gel after a PCR run". To answer your question, we have to know where this contamination come from. High absorbance at 230 nm may originate from carbohydrates (thank you, Tewodros) and some organic solvents used for DNA extraction. The thing is that they cover a broad absorbance spectrum and may absorb even at 260 nm, masking the absorbance peak of nucleic acids. So you may mistakenly conclude you have a high DNA abundance in a sample. Moreover, some carbohydrates, phenolics and organic solvents may impede PCR reaction. 

Hi! What are the values acceptable for this ratio? Thank you..

i have processed stool sample for dna extraction by both the methods, spin column as well as PCI extraction, and in both cases, i got less 260/230 ratio, and slightly high 260/280 ratio. PCR as such failed for amplification. can anyone guide for troubleshooting.???

I have realised over the time that during RNA isolation from samples, homogenisation of sample is one of the crucial step for good quality RNA. After homogenization for 1-2 min, depending on toughness of sample, 3-5 min of additional incubation of Trizol-tissue homogenate at RT is advantageous. Low OD 260/280 ration is not an issue, in majority of the samples.

Ratan

As said in the post before, washing our RNA pellet twice with 1 ml ice cold 70% EtOH has definitely increased the purity of the RNAs we use for our RTs (360:230 up to 2.5)

What if the 260/230 ratio is a negativr value? What does that imply?

Has never happened to me, but my guess would be something is wrong would be that you either didn't use the solvent that you had your samples in for the blank or that your solvent is contaminated.

Hi everyone

I sent the PCR products for sequencing (forward and reverse primers separately). The result of the Sanger sequencing was so clean with high quality for forward sequence but I don't know what happened for reverse sequence. That was completely disappointing. What can be the cause of failed DNA sequencing by reverse primer?

The PCR products were purified by purification kit protocol, the 260/280 and 260/230 ratios were 1.8 and 1.25 respectively and the NA concentration was 50.4 ng/ul. 15 ul cocktails (12ul PCR product +3 ul of each primer) is prepared for sequencing.

The chromatogram of forward and reverse (reverse complement) sequences have been attached.

thank you in advanced