How important is the 260/230nm ratios for squencing purposes?

[strawbaubz]

Hi all

I was wondering if anyone could give me some input. I am doing ampicon sequencing on the Miseq illumina platform. I am doing 2 X 300 bp paired end reads. I am using universal 16S primers that have been adapted with the adapter sequences required by the illumina Miseq platform. When I run a PCR I get the right fragment lenghts but a lot of non-specific band also of larger size. Due to this I am forced to extract the correct sized band from the gel and do a gel clean-up. I have now tried two different gel extraction kits and although the DNA comes out pure (good 280/260nm ratios) my 260/230nm ratios are extremely low. I have tried many different ways to optimize this by increasing the number of wash steps and discarding the flow through and doing an additional dry spin thereafter. I have used a low percentage gel of 0.8% and also run a very thin gel. But nothing has proved to increase the ratio 260/230nm.

My question is how important is this ratio for NGS??? Does anyone experience this and have some simple tricks to better the ratio. I have tried the zymo gel extraction kit and the Qiagen mini elute kits. Both give the same very low ratios. Will sequencing be hampered by this low ratio if the DNA purity ratio 280/260 is fine???? Help would be greatly appreciated. I have run out of ideas.

[microgirl123]

I'd really hesitate to run samples with a bad 260/230 ratio. We do a lot of Sanger sequencing, two-step PCR library prep, and genomic DNA library prep and anything with poor 260/230 ratios does not sequence well, if at all, and the library prep does not work. I generally tell our users that gel extraction needs to be avoided at all costs. Lots of things that inhibit reactions absorb at 230! If possibly, I'd order just locus-specific primers (because they are cheap) and try to optimize your PCR with those to remove the non-specific bands.

The 260/280 ratio is measuring your DNA to protein amounts. With the kits used to extract now DNA this ratio is usually good. I think it had more significance in the past when phenol-chloroform was used to extract DNA.