N.B.: Urea takes time to dissolve and may require gentle warming (40-50°C) so try to prepare the two urea containing solutions in advance.
solutions to prepare
- 10% w/v ammonium persulfate in water (can be stored at 4°C)
- 100 mL 20% acrylamide/urea solution:
49.85 g UREA
0.5 g N,N methylene bisacrylamide
50 mL acrylamide 40% stock (Bio-Rad 161-0140)
10 mL TBE 10X
- TBE 10X :
108 g Tris base
56 g boric acid
40 mL 0.5 M EDTA pH8.0
double distilled H2O (ddH2O) to 1L
- 100 mL of TBE/UREA solution:
dissolve 49.85 g UREA in 80 mL autoclaved H2O
add 10 mL 10X TBE
top up to 100 mL with autoclaved H2O
- formamide loading buffer:
10 mL formamide
10 mg xylene cyanol FF
10 mg bromophenol blue
200 µL 0.5 M EDTA pH8.0
Casting and pouring the gel
Choose the adapted % for your transcript :
dye migration according to denaturing acrylamide %
![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgTm-EfdLfQCJBCMsnIktgvmxzmKTPDq6NRN0WadKLerKDHWw8cmJ16tUJq8fLRZ5COGt4cK2qQBxY3oPk6Im6PnYnHme0tBC3u8qevXk3LtGKU9IahlJqAlB3ySh4s6hiphitzDWTuBO0/s400/chart1.jpg)
Then cast a 1 mm x 20 cm x 20 cm gel, taking care to clean both glass plates an d silanizing one of the two plates (silanizing liquid available from Lonza).
- gel recipe:
X mL acrylamide/UREA8.3M
TBE/Urea to 40 mL final
240 µL 10% ammonium persulfate
50 µL TEMED
This gel polymerizes very fast due to the presence of urea, be careful !!!!!
Avoid leakage by pouring the gel as horizontally as possible, bubble formation should be avoided but if bubbles appear, you can always try to get rid of them by playing with the left/right inclination of the assembled glass mold.
clean the wells with a syringe equipped with a bent needle.
pre-run the gel in TBE 1 X for 30-60 min at 500V
Sample preparation
During the pre-run, add 40 µL of loading buffer to the 50 µL transcription product redissolved in EDTA and heat 5 minutes at 95°C,
chilled on ice,
stop the pre-run f the gel,
clean the wells a second time, with a syringe equipped with a bent needle prior to load,
load the samples carefully and run the gel keeping the same conditions as in the pre-run.
Monitor RNA
RNA should be accurately visualized by UV shadowing : using a UV lamp (I used 254 nm to better see the RNA, but higher lambdas are considered less damaging for RNA) and a silica coated plastic sheet (usually used in Thin layer chromatography).
Uncast the gel when the run is over and dispose it on a cellophane film, put the silica sheet under this cellophane and directly expose the gel to the UV source, which gives this from top to bottom:
UV light
gel
cellophane
Alternately, if the RNA was transcribed with radioactive alpha 32P UTP, you just have to monitor its presence by autoradiography.
Cut the band of interest, chop it in very small slices.
Elute it in elution buffer:
elution buffer can vary depending on the type of experiment that follows the purification.
If you want to 5'end label the RNA for instance it is advised not to use tRNA in the elution buffer, whereas this is not a problem for RNA that has been body labelled with radioactive UTP during transcription.
For 5' end labeling it is also important NOT TO USE DEPC anywhere since this compound may inhibit dephosphorylation and phosphorylation steps.
elution buffer with tRNA (400 µL should be sufficient) :
0.75M ammonium acetate
0.1% SDS
10 mM MgOAcetate
0.1 mM EDTA pH8.0
add at the last minute
25 ng/µL bovine or E.coli tRNA
10 µL/mL phenol buffered with 0.1M citrate pH 4.3
elution buffer without tRNA (1 mL should be sufficient):
50 mM Tris-HCl pH7.5
300 mM NaOAcetate
0.5% SDS
Incubation can vary but minimum is of 2 hrs at room temperature (tried with no tRNA) or 37°C (tried with elution buffer with tRNA and phenol) under vigorous shaking.
Once elution is performed, treat with acidic phenol, chloroform and precipitate overnight with NaOAc and pre-chilled absolute ethanol.
Avoid using ammonium acetate if the RNA is prepared to be used for radioactive 5' end labeling.
resuspend RNA in autoclaved water.
hi
ReplyDeletethank you so much for the post!
ReplyDeleteThank you for contributing. I wasn't expecting any reply to be honest. But now that I saw yours it feels great to be useful to someone.
ReplyDeleteHI Nico, could you please let me know what kind of gel system you used? I only have QC gel system, but not for prep gel. Thanks! - Lois
ReplyDeleteDear Lois,
Delete- concerning the casting apparatus:
I used custom made high temperature resistant glass plates (5 mm thick x 20 cm x 30 cm) with 1 mm thick teflon spacers.
- The running power supply was an old one from BIO-RAD.
These old power supplies deliver higher voltage than the newer ones and I believe are less prone to sudden crash upon extended period of use.
- concerning the running tank: I used a custom-made tank (done by university workshop).
Concerning the gel mixture itself, I used a denaturing UREA PAGE with something like 8M urea final in the gel.
The running buffer was a 1x Tris- Borate-EDTA buffer.
Overall, you can do this kind of run in any gel format. The only important thing is that resolution of your bands will decrease with the size of your gel (unless you do a special kind of gradient denaturing gel) and the increase of your sample volume ( the thinner the samples, the least blobbish are the bands on the gel; wide wells help a lot here).
The heating of the gel due to the Joule effect originating from the application of high voltage to a big gel (i.e. a strong resistor) is normal and also contributes to keeping the RNA in a denatured state, even though you should avoid to cook the poor thing integrally.
Since this was a preparative experiment in my case (purification before immobilisation) , I had big samples and needed big wells to load them in and further have clean flat lanes to monitor and cut.
I hope this detailed answer could help you.
Good luck!
Nico
Thanks Nico for your detailed reply! very much appreciated! We use the similar system for DNA PAGE purification, normally loading 50 to 100 OD. However, IVT RNA normally can't get that much yield. So I think I need a smalled size gel. You gave me some idea though. Thanks for share your experience and techniques! - Lois
ReplyDeleteThank you for this extremely brilliant blog! We really appreciate your blog post. There are actually a multitude of techniques we could put in to make really a good use of information without much efforts and financial resources. Thank you so much for giving light to many problems we haven’t come across before using your blog.
ReplyDeleteRNA Purification Kits
Hi, can I know, what is the purpose of the silica to visualize the gel, instead why cant we just visual the RNA on normal surfaces?
ReplyDeleteThank you.