Hello dear researchers:
I am perfoming DNA extractions from a Gram-positive bacteria using QIAGEN DNA mini kit. For standard amount of bacteria (picked up from plate) resuspended in PBS, I obtain concentrations between 80 and 300 ng/ul, A260/A230 between 1.7 and 2.1, and A260/A230 between 0.9 and 1.7 after following manufacturer´s instructions. Is there a way to improve A260/A230 parameter?. I am going to perform whole-genome sequencing using Illumina tech, is this parameter important?
Best regards,
Adrián
The 260/230 & 260/280 ratios indicate the purity of your DNA. Generally, pure DNA should have a 260/230 absorbance ratio of roughly 2.0-2.2, and a 260/280 of about 1.8. If it's a little less than that, it's usually not a problem. If the 260/280 ratio is appreciably lower, it may indicate he presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. Likewise, if the 260/230 ratio is considerably lower than 2.0, then this indicates contaminant that absorb at 230 nm.
I am not sure which of the ratios you mentioned are for 260/230 and which for 260/280--you mentioned "A260/A230" twice. I think the 0.9 is a bit low, but any values of 1.7 or higher should be fine (regardless of which absorbance ratio you are talking about here.) To be sure you might want to discuss this with the staff of the facility that does the Illumina sequencing--they might be able to let you know which ratios are acceptable.
If it turns out you really need to improve the purity of your samples, have a look at the Qiagen handbook/protocol. It has some good tips for that. It lists the following reasons/recommendations if the ratios are too low:
"A260/A280 ratio for purified nucleic acids is low
a) Inefficient cell lysis due to insufficient mixing with Buffer AL: Repeat the procedure with a new sample. Be sure to mix the sample and Buffer AL immediately and thoroughly by pulse vortexing.
b) Inefficient cell lysis due to decreased protease activity: Repeat the DNA purification procedure with a new sample and with freshly prepared QIAGEN Protease stock solution. Be sure to store the stock solution at 2–8°C immediately after use. Ensure that QIAGEN Protease is not added directly to Buffer AL.
c) Inefficient cell lysis or protein degradation in Buffer AL or Buffer ATL due to insufficient incubation time: Repeat the procedure with a new sample. Extend the incubation time. Take care that no residual particulates are visible (bones or hair will not be lysed at all).
d) No ethanol added to the lysate before loading onto the QIAamp Mini column: Repeat the purification procedure with a new sample.
e) Low percentage ethanol used instead of 100%: Repeat the purification procedure with a new sample
f) Buffer AW1 or AW2 prepared incorrectly: Check that Buffer AW1 and AW2 concentrates were diluted with the correct volumes of pure ethanol (see page 16). Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone. Repeat the purification procedure with a new sample."
If this doesn't work for you, you can try various DNA cleanup methods.
I hope this helps.
Cheers, Oliver
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