7 replies to this topic

[vitalgene]

Hi all,
Unfortunately the RNA which I eluted is too dilute for my further steps, my question is that can I use a vacuum centrifuge for concentrating it, if so what should be the parameters, secondly we have an in-house Beckman vacuum optimaL60 ultracentrifuge can this be applied to concentrate the samples
Thanks in advance

[Kristina @Invitrogen Dynal]

Hi

Why not try Dynabeads mRNA Purification kit? http://products.invitrogen.com/ivgn/en/US/...productID=61006

Beads with oligo d(T) will bind to the mRNA in your sample and you can elute in a small volume to increase the concentration.

Kristina

[mdfenko]

vitalgene, on Sep 4 2009, 04:44 AM, said:

Hi all,
Unfortunately the RNA which I eluted is too dilute for my further steps, my question is that can I use a vacuum centrifuge for concentrating it, if so what should be the parameters, secondly we have an in-house Beckman vacuum optimaL60 ultracentrifuge can this be applied to concentrate the samples
Thanks in advance

the vacuum in the ultracentrifuge is for the purpose of reducing heating of the rotor by friction with the air in the chamber. it does not serve the same purpose as the vacuum in a vacuum concentrator system (like a speedvac).

using a speedvac (or equivalent system) will concentrate everything in the solution. if that won't affect your later procedures then, by all means, try it. if it will affect later then try either something like recommended by kristina... or by reprecipitating and reconstituting in lower volume.

[molgen]

If your lab is low on funds you can do one of the oldies but goodies instead of a column or magnetic beads.
An ethanol precipitation or glycogen will do the trick just as good.

[cupidstunt]

suggestions above are all good. just to add, we sometimes use amicon ultra centrifugal filter devices. (Millipore)

[wntiong]

cupidstunt, on Sep 24 2009, 10:13 PM, said:

suggestions above are all good. just to add, we sometimes use amicon ultra centrifugal filter devices. (Millipore)

I have the same question - my microarray work requires only 10 ul of rna to proceed but i have about 30 ul of rna (after spin column), can i use vacuum concentrator to reduce the volume to 10 ul? I dare not to try ethanol precipitation because everytime i tried, many rna gone.

[moerae]

wntiong, on Oct 14 2009, 08:22 PM, said:

cupidstunt, on Sep 24 2009, 10:13 PM, said:

suggestions above are all good. just to add, we sometimes use amicon ultra centrifugal filter devices. (Millipore)

I have the same question - my microarray work requires only 10 ul of rna to proceed but i have about 30 ul of rna (after spin column), can i use vacuum concentrator to reduce the volume to 10 ul? I dare not to try ethanol precipitation because everytime i tried, many rna gone.

Personally I wouldn't use speedvac or something similar just because I wouldn't know what's been used in our machines prior, but with ethanol precipitation adding glycogen really helps, and it's always reassuring to see a pellet there at the bottom of the tube.....

[Helios]

i have used speedvac to concentrate my rna samples for microarray expts.the labeling was fine with the concentrated samples.
just make sure you clean the speed vac to avoid rnase contamination