Hi,
I have some bacterial genomic DNA extracted using the QIAgen QIAextractor. When I ran it through a gel, it also had RNA in it.
This is new as we have just had our extractor upgraded, and something about the new reagents now allows RNA through. Can I treat the already extracted samples with RNase A rather than re-extracting and adding it to my lysis step?
They are going for whole genome sequencing. I don't mind redoing them, but it would be better if I didn't have to!
Any ideas would be great.
So this is a very old post, and I am sure that you are way past it Hayley! Nevertheless, since others may stumble upon it as well, I thought I'd add some important info. I have learnt this the hard way, and got good help from others in here.
Short version: Yes, you can, BUT BOIL THE RNASE A BEFORE USE!
Longer version: According to most manufacturers, you should be able to perform an RNase A treatment to previously extracted DNA, and according to most manufacturers their RNase A should be DNase-free. However, DNase copurifies with RNase, and even in the supposedly DNase-free RNase A available, there are often trace amounts of DNase present. And that's all it takes... I've ruined precious samples, which degraded badly when I treated them according to the manufacturer's instructions. This is apparently not a provider-specific problem, instead people have had this experience with several brands.
Luckily, there is a simple solution, which I nowadays apply even when performing the RNase treatment during the DNA extraction – when there is much lower risk for DNA degradation. RNase A is very thermostable, unlike DNase. Thus, before use, boil the RNase for ten minutes and let it cool. Centrifuge, and if there is a pellet, remove the supernatant (the cleaned RNase A) into another tube.
On a side note, in many protocols where RNase A treatment is prescribed, I don't think they are necessary, as long as you use a reliable (fluorometry-based) quantification method that does not risk co-quantifying RNA when you quantify your DNA (which usually happens with spectrophotometers).
Good luck everyone, and let's not ruin more DNA samples now! Cheers,
Martin
Hi Hayley, go for the RNAse treatment, DNA should not be affected.
Cristina
Thank you Cristina
I haven't had to do this before....do you know what I need to do? DNA currently in 96 well boxes approx. 200ul of elution
Hi Hayley,
To remove your RNA contamination you can follow the link of promega kit to extract DNA from various sources and removing contaminating RNA (in 96-well systems too). If you apply RNAse A to your samples and follow the methods, i guess it should work.
All the best.
https://norgenbiotek.com/product_resources/bacterial_genomic_dna_isolation_kit_bacterial_genomic_dna_isolation_96well_kit__protocol_no_barcode_17900_2083.pdf
Adding RNAase to the sample followed by precipitation should remove the RNA. This should work instead of re-isolating the samples.
Hi Hayley,
Did RNAse treatment work for you? I have heard that there is a risk of losing a lot of the DNA as well.
cheers
David
So this is a very old post, and I am sure that you are way past it Hayley! Nevertheless, since others may stumble upon it as well, I thought I'd add some important info. I have learnt this the hard way, and got good help from others in here.
Short version: Yes, you can, BUT BOIL THE RNASE A BEFORE USE!
Longer version: According to most manufacturers, you should be able to perform an RNase A treatment to previously extracted DNA, and according to most manufacturers their RNase A should be DNase-free. However, DNase copurifies with RNase, and even in the supposedly DNase-free RNase A available, there are often trace amounts of DNase present. And that's all it takes... I've ruined precious samples, which degraded badly when I treated them according to the manufacturer's instructions. This is apparently not a provider-specific problem, instead people have had this experience with several brands.
Luckily, there is a simple solution, which I nowadays apply even when performing the RNase treatment during the DNA extraction – when there is much lower risk for DNA degradation. RNase A is very thermostable, unlike DNase. Thus, before use, boil the RNase for ten minutes and let it cool. Centrifuge, and if there is a pellet, remove the supernatant (the cleaned RNase A) into another tube.
On a side note, in many protocols where RNase A treatment is prescribed, I don't think they are necessary, as long as you use a reliable (fluorometry-based) quantification method that does not risk co-quantifying RNA when you quantify your DNA (which usually happens with spectrophotometers).
Good luck everyone, and let's not ruin more DNA samples now! Cheers,
Martin
To avoid any risk, divide your samples in two equal vol., add RNase A for one. Then you can check the purity and quantity of RNase A treated sample with non-treated one by gel electrophoresis. Good luck.
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