Hi, I need to sequence, whole genome, of a bacterium (Burkholderia species) using PacBio. However, it turns out (based on what the supplier says) that PacBio is very sensitive to RNA contamination and DNA degradation, meaning they are unable to proceed if the DNA sample is contaminated by RNA to certain levels. I extracted the DNA using several spin column kits (Qiagen, Promega, MoBio) but I always get RNA although I always included RNase, even doubled the concentration and time. Can anybody suggest what method can I use to overcome this? I know that bacteria are very active and produce a lot of RNA. Thanks.
If you have your DNA ready, treat with RNAse as mentioned by Sana, to remove that RNAse treat/spin with Chloroform: isoamyl alcohol and reprecipitate your DNA.
or
If you want to isolate DNA again add RNAse (and incubate as per the procedure) in one of the steps before going for Chloroform: isoamyl alcohol then you need not do it again and RNAse added also be removed automatically.
Mathew A Beale Hi Mathew, thanks for the response. I detected the RNA on gel when I run the DNA, which sit at the bottom of the gel. And based on the supplier's QC, the intensity of the bottom band (could be RNA) was prohibitive for PacBio sequencing. Since you mentioned that depleting RNA may not be useful, can I treat the isolated DNA with RNase and then re-precipitate the DNA? In my work I added RNase treatment at the early step (extraction) according to manufacturer's instruction. Cheers.
I don’t have PacBio expertise but if the treatment with RNase is ineffective you could try an enrichment by pre-sequencing step by PCR, using one or more primers designed all along the target genome
best
Hi Pak Cahya, I did the HMW DNA extraction from fungal tissue in the past. Maybe my protocol can help. dx.doi.org/10.17504/protocols.io.9g3h3yn
Cheers,
Rey
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