Alright, here you go - with the following caveats:
1) These protocols are all written for people doing this form scratch. If you end up going with pre-cast gels, you can probably ignore 80-90% of this. Handy recipes for DIY sample loading buffer, pictures and step-by-step instructions for PAGE gel casting, etc. - good things to know, whether you'll actually be doing the work or not.
2) 2 of 3 protocols are for people doing RNA purification using PAGE - also something you can safely ignore. Everything up to the sample excision from the gel will be identical.
3) The last .pdf is pretty big, and it free access, so grab it here:
As for using pre-cast gels, I'm only familiar with the systems I already mentioned (Bio-Rad's Mini Protean rig and Invitrogen's XCell Mini), both of which are available fairly cheaply on eBay, and in my opinion basically equivalent. They are obviously not compatible with other brands of power supply, so that's probably worth keeping in mind. Also - last but not least - while you will save yourself a ton of trouble by doing the pre-cast thing, you will get better resolution on a longer gel, so that's the price of convenience.
I forgot to mention markers, by the way. NEB makes a microRNA marker that should work well for your size range:
On Sun, Feb 12, 2017 at 2:04 PM, Mega [Andreas Stuermer] <masterstorm123@gmail.com> wrote:
Awesome, thx for the info! Protocols would be highly appreciated of course--
On Sunday, February 12, 2017 at 7:21:25 PM UTC+1, Rikke wrote:Hey Mega,
I work with small RNAs as a major part of my dayjob, and I can tell you from first-hand experience that agarose will not be able to provide the kind of resolution you're looking for. You'll need to be using a high-percentage denaturing PAGE gel - preferable 15-20% TBE-Urea.
You can minimize your exposure either by using pre-cast gels - if you have access to a compatible rig (Bio-Rad, Invitrogen, possibly others - look around on eBay for a used gel rig) - or pre-mixed reagents for casting your own. The second option obviously requires a lot more gear, and casting PAGE gels is quite a bit more finicky than pouring agarose gels. Either way, keep in mind you'll need a denaturing loading buffer, a way to stain your RNA (e.g. SYBR Green II), and a way to safely dispose of everything afterwards.
I can dig up some manuals / protocols at work tomorrow if you need more info.
On Feb 10, 2017 3:23 AM, "Mega [Andreas Stuermer]" <masters...@gmail.com> wrote:Hi, I was wondering if anyone has worked with small RNAs (20-50 bp) in a DIY setting. I heard you can't visualize them on an Agarose gel well. No chance you can distunguish 22 bp and 20 bp?
Ideally some method without toxic ingredients ;)
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