RNA gel electrophoresis - any recommendations? - (Jul/30/2007 )
I am going to analyze an in vitro transcription using a denaturing RNA gel. Preferably, I would like to use a kit for this, i.e. Elementary RNA gel kit (amresco-inc.com/catalog/PDF/Q1%202006%20INTL.pdf). Have anyone tried out this kit and have any possible recommendations?
I have never tried the Elementary kit, and I have also seen other kits available searching the web.
All recommendations and tips are highly appreciated!
-Cathrine:)
we made the RNA gels ourselves, never bought it.
If you need, I can send you my gel recipe. You could also search the forum, I think it was discussed here before.
Oh...its very easy to make RNA gels and then run it. In fact, I observed that it is quite easy to even run and see RNA bands on a DNA gel. Otherwise even the RNA gels are quite easy to prepare just that you have to use formaldehyde!!
If you need, I can send you my gel recipe. You could also search the forum, I think it was discussed here before.
How much time does it take to make one? Do you make all the ingredients by scratch?
Do you think it would work out to use a standard agarose DNA gel, but mix my RNA sample with RNA loading buffer, as sold by different manufacturers?
The recipies for making gels on my own that I have seen seem to be so time-consuming, considering that you have to make EDTA, MOPS, loading buffer, sample buffer, deionize formamide and so on. I have quite a little time at the moment, so it would be great to save some time..
for in vitro transcription assays, i use a 10% 8M urea gel. Im' dealing with VA1 gene.
for 25 ml t's 10g urea, 5ml acryl 29:1 40% and 5ml TBE 5X. I vortex till urea dissolves (5')
then 250µl aps and 25µl temed
so the time for pouring it...... let say 20'
Thank you for all advices!
This is the protocol that I had planned to carry out ( http://www.promega.com/guides/rna_guide/workingwithrna.pdf , page 4). But it seems so time consuming.
Have any of you tried it out?
I buy the invitrogen TBE Urea gel with Novex® TBE Running Buffer (5X). also with the Novex® TBE-Urea Sample Buffer (2X)
the gel comes in 10, 12 and 15% depending on the size of your transcript.
it's really easy to use. but you'll probably need their running apparatus.
detect with SYBR® Green II RNA gel stain is also super easy. which then you can just look vitualize with your regular gel box.
ok so you plan to do agarose denaturing gel...
Well Mops is quite tough to prepare.
The point is that you need to melt the agarose and prepare water bath at 55°.
you need to allow the agarose to cool in this water bath. generally i let 15 20' and then agarose start to gelify. Just agitate the becher is ok.
then add formamide/formaldehyde and pour immedately under vaccum hood (formaldehyde and formamide vapors are toxic). Let solidify.
So at this step it will take 1h.
during solidification of gel, prepare samples.
The running takes roughly 2h at 80-100v under vaccum hood and check for heating of the buffer (i start at 100V, but if it's too hot i decrease to 80)
if you want to capillary, transfert the gel, prepare bunch of paper during the running. Note : It's quite long to prepare ! as you need at least 5cm of paper when pressure is applied!
I cathrief I checked the protocol that you are going to use. Believe me its very easy and not as fred has written. Once you make MOPS buffer (make quite a lot of it so that you dont have to make everytime) you will find that the rest of it is very easy. If you have enough RNA, why dont you run one DNA gel and load your RNA samples, you will sure see the RNA bands.