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alqga

Member Since 14 Aug 2012
Offline Last Active Oct 05 2012 01:23 AM
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Topics I've Started

Low 260/230 ratio depending on tissue.

23 August 2012 - 09:55 AM

Hello,

I am extracting RNA from different tissues (liver, spleen, brain) and also of isolated immune cells from head kidney and blood with the Trizol method. With liver and spleen I got really good 260/280 and 260/230 ratios but when I extract RNA from the brain and from the isolated cells I got good 260/280 ratios but very low 260/230. I have performed the process at the same time so I don't know why there are such huge differences between tissues. I read that in some tissues is just more difficult to extract pure RNA, so my second question is, how important is this 260/230 ratio for qPCR?

Thanks!

same primers, different product in conventional PCR and qPCR

14 August 2012 - 12:23 PM

Hello,

I am new here and I don´t know if this question has already been answered but here it goes.

I have run a qPCR with new primers and I got amplification and nice melting curves. IN addition I loaded an agarose gel and the product had the expected size (around 180 bp)

However, I wanted to sequence the product to be sure and when I run the conventional PCR  prior to the purification of the DNA and I loaded a gel to see the size of the product I obtain a huuuge product (1000 bp) what has nothing to do with the product I got in the qPCR

Any hint about what can be?? (sorry if this is too basic but I´m quite new...)

Thanks!

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