Hi Oliver

Lower ration of 260/230 could be due to the presence of salt or carbohydrates carry over which happened to me when I was extracting DNA from plants. Ethanol precipitation did not help as well. I had to use DNA columns to improve purity.

I guess you should use columns as well.

Best regards

Hi Ali,

Thanks for your answer. I'm reluctant to use columns for this as (to my knowledge) they don't recover fragments of the length I'm looking for. Have you been recovering fragments >10kb with columns?

Hi Oliver

If you consider DNA extraction, I would agree with you but you have extracted DNA, only you need to purify them with much lower G-force and time for centrifuge. It should work then.

• Agreed: large DNA constructs can get stuck in columns and fail to elute fully
• I have looked at your DNA extraction method and extrapolating from my own genomic extractions I would contend that your low 260/230 ratios are due to insufficient removal of salt and in particular sodium chloride
• Accordingly, I would suggest the following :
• 1. After RNAse digestion, Perform a chloroform extraction and then remove upper aqueous layer; to which you should add your sodium chloride and ethanol
• 2. Having precipitated RNA wash 2 x with ROOM TEMP 70% ethanol before precipitating your RNA and re suspending finally in TE
• As some body who has performed many genomic extractions with various tissue types I have to say your method is crude and in the absence of a phenol chloroform extraction and/or column clean up you are likely to have excess protein (even with Prot K digestion), thus reducing your 260/280 and an excess of salt with no obvious wash; thus reducing your 260/230
• 3. Alternatively, instead of adding sodium chloride to the RNASe digested lysate you would simply add an equal volume of isopropanol and 2.5 to 3 volumes of cold ethanol before spinning and washing with 70% ethanol at room temp as described. This alternative alcohol ppt is instead of 1. and 2. above
• Try both and see which works best in terms of purity and recovery

Hi,

I agree with Laurence Stuart Dawkins-Hall .

You can try this protocol for DNA purfication after salting out:

https://www.thermofisher.com/br/en/home/references/protocols/nucleic-acid-purification-and-analysis/dna-extraction-protocols/phenol-chloroform-extraction.html

Remember to use pH slightly alcaline in this extraction, do not use glycogen, i think you have a lot carbohydrates in your preparation.

Good luck,

Jacó

Hi,

here is some useful material:

https://www.researchgate.net/post/What_would_be_the_best_method_to_increase_260_230_ratio_of_genomic_DNA

https://www.researchgate.net/post/How_can_I_improve_RNA_quality_with_low_260_230_ratio

https://www.researchgate.net/post/How_to_improve_my_260_230_ratio_for_DNA_high_purity

https://www.researchgate.net/post/What_is_the_effect_of_a_low_260_230_ratio_on_the_purity_of_DNA

Best regards