RNA denaturing gels

Hello Experts,

I am trying to run a RNA gel, (which I had run a very long time ago, and
time tends to erode all information from my chamaeleonic nervous system!),
ergo, here are the details:
I make a mRNA by the mMessage Machine kit from Ambion. The mRNA is capped,
with a poly A tail of about 19 bases. The insert size is ~2kb, and so I
expect a band around 1.9-2.1 kb.
When I regular agarose gel, I see some band ~1kb. I have two other inserts
about the same size, and I do'nt really see much with them.
I tried running a folmaldehyde gel, and the protocol I followed had a tonne
of EtBr in the sample buffer, which ofcourse runs in the opposite direction,
and gave me a large background. I allowed it to run out of the gel.... then
the bands I got were diffuse, and I could'nt really say what size. The
marker was another story: this one from NEB, which did'nt show up at all.
I obviously don't know too much about RNA gels, and if anyone could shed
some light on how to better the technique or interpret results or even
anything on the Ambion message kit, I'd appreciate it much.

thank you,
pow

Hi Pow,

We have used the RNA ladder from Ambion with no problem. But all our RNA
work is done on an Agilent Bioanalyser. I don't have much experience with
gels but I'm under the impression that EtBr binds less well to single strand
vs double strand. So the protocol's use of a tonne of EtBr may have some
basis. I would suggest two things, using a control plasmid (from the kit if
Ambion supply one) and try to silver stain your gels rather than use EtBr. I
don't know if there is a more RNA friendly fluorescent dye to use in place
of EtBr, anyone?

Ian Mc

"Pow Joshi" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ] .net...

SYBR gold is excellent for detecting both single stranded and double
stranded DNA and RNA. I do a 20 minute soak of gel running buffer, 50%
glycerol and 1-0.5x SYBR gold and get strong visualization of single
stranded DNA.

On 9/24/07, IanMc <[Only registered users see links. ]> wrote:

There's also Sybr Green II RNA Gel Stain from Invitrogen; Ex/Em: 254,
497/520 nm, bound to nucleic acid Here's a couple of links:
Product note:
[Only registered users see links. ]
Some tips on using Sybr stains:
[Only registered users see links. ]

>> > Hello Experts,

formaldeyde gels are known to give fuzzy bands, but is often used because
you can do northern blots with the gel afterwards. If your simply wanting to
size your RNA then I suggest you run a gloxal gel, which will give you a
cleaner band. In both cases, you need to make sure all your solutions are
RNase free and your gel tank and casting comb has been washed with a
solution to kill RNases (e.g., alcoholic KOH) and then with RNase free water
to restore PH. Goodluck.

Thank you everyone for the input. I shall try the new methods for my RNA.

Pow

On 9/25/07, corona <[Only registered users see links. ]> wrote:

Pow,
I hope u have run the "new" RNA gel by now. I want to share few thing with
you.
I use RNA for RTPCR so i just need to check for the integrity. I dont use
denaturing gels and so i use regular DNA agarose gel (TAE buffer,
2microLEtBr) and i get 3 expected bands including 5s one.
So depending on why u need a RNA gel, u can go for either denaturing and all
those excercises or stick to regular DNA gel..
Hope it helps.

Sudheendra.


On 9/26/07, Pow Joshi <[Only registered users see links. ]> wrote:



--
Think before agree
Think before you nod
but STOP thinking
and You Are God

On 10/17/07, Sudheendra Rao N R <sudhee26@gmail.com> wrote:


Thanks Sudheendra...... I have usd regular gels, however, I wish to
ascertain the integrity of mRNA, which I make using the Ambion kit. The
regular agarose gels seem to have some issues with the size assessment since
the RNa would have some scondary structure. I have now resorted to using the
Agilent analyser, and am hoping I can make some sense of that data
cheers
Pow



Sudheendra.