The best method for recovering RNA fragments from poly(lactonamide) gels is the crush-and-soak method, which provides excellent recovery of high-purity single-stranded RNA from denaturing gels. This method provides good recovery of high purity single-stranded RNA from denaturing gel plants, but it is not suitable for long transcripts because it is time consuming and inefficient, e.g. less than 30% for RNAs larger than 3kb. However, this method is very effective in recovering small fragments of RNA.
Operation method
Recovery of RNA fragments from polyacrylamide gels
Materials and Instruments
Ethidium bromide Elution buffer Stock solution 8mol LLiCl solution Isopropyl alcohol 70% Ethanol 1XTBE Buffer Move I Materials and equipment Caveat 1) When placing Whatman 3MM filter paper on the gel surface, be careful to prevent bubbles and wrinkles.2) When handling radiolabeled fragments. Be careful to prevent radioactive damage. For more product details, please visit Aladdin Scientific website.
Polyacrylamide gel electrophoresis tank Metal spatula Whatman3 MM Filter paper Glass rod Surface dish Cling film Transmission UV lamp Blade Texta pen Radiographic film
1) Ethidium bromide: 10 mg/ml aqueous solution
2) Elution buffer: 80% formamide dissolved in 40 mmol/LPIPES, lmmol/LDTA, and 400 mmol/LNaCl (l0 mmol/LDTT if 35S-labeled RNA is to be recovered).
3) Stock solution: 1Ommol/LDTT in sterile water.
4) 8mol/LLiCl solution
5) Isopropyl alcohol
6) 70% ethanol
7) 1XTBE buffer
8) Polyacrylamide gel electrophoresis tanks
9) Metal spatula
10) Whatman3 MM filter paper
11) Glass rod
12) Surface Dish
13) Cling Film
14) Transmission UV Lamp
15) Blade
16) Texta pen
17) Radiographic film.
II Methods of Operation
(I) Preparation
Perform polyacrylamide gel electrophoresis on the RNA samples.
(ii) Recovery of destination RNA fragments
1. Recovery of non-radiolabeled RNA fragments
1) Remove the gel plate from the electrophoresis tank and place it flat on a table. If only the front plate is siliconed, place the concave back plate face up before separating the two plates. After separating the two plates, the gel should still be stuck to the front plate. Insert a small metal spatula between the two plates and carefully pry them apart. If the gel sticks to the G plate, turn the plate over and try again. In any case, the gel should still end up sticking to one of the glass plates as a support.
2) After the two glass plates have been separated, place the gel on a piece of Whatman 3 MM filter paper. Place the glass plate horizontally with the gel facing upwards, then place a piece of pre-moistened Whatman filter paper 2-3 cm larger than the gel on the gel surface, and gently roll the glass rod over the surface of the filter paper to remove air bubbles and wrinkles and to make the filter paper stick flatly to the gel to remove the corner of the filter paper. Both are then removed from the glass plate. The paper and gel should be removed at one time, and care should be taken to be gentle. Then immerse both in a shallow surface dish containing 0.5 mg/ml ethidium bromide dissolved in 1XTBE and stain for 15 to 45 min before removing the gel and filter paper.
3) Place a piece of good-quality plastic wrap on the surface of the UV-transmitting lamp, place the gel side on the surface, and gently peel off the Whatman filter paper. The gel can be viewed under transmitted UV light.
4) Determine the target RNA band, then cut the target band from the plastic wrap and place it in a centrifuge tube.
5) Squeeze the gel block with a pipette tip to crush the gel. Cover the gel with 1 to 2 times the volume of elution buffer, up to 0.5 ml, and incubate at 37 ℃ while gently shaking the gel to facilitate the elution of RNA. fragments smaller than 500bp will be eluted within 3 to 4 h. Larger fragments may take about a day to elute. Larger fragments may take about a day to elute.
6) Centrifuge, recover the supernatant, rinse the gel with 0.5 volume of elution buffer, centrifuge, recover the supernatant, and combine the supernatants.
7) Precipitate RNA with double the volume of isonitol, centrifuge to recover and since RNA recovery of small fragments is very complete, there is no need to add an RNA carrier here.
8) Wash the RNA precipitate with 70% ethanol and resuspend the RNA with 10 mmol/L DTT (preferably with an RNAase inhibitor if possible). re-precipitate the RNA by adding 0.1 v/v of 8 mol/L LiCl and an equal volume of isopropanol.
9) Centrifuge the model to collect the RNA and wash the RNA precipitate with 70% ethanol.
10) Dissolve the RNA sample in 10 mmol/LDTT and store at 70 °C.
1 Recovery of radiolabeled RNA fragments
1) Pry apart the two plates as described above and cover the gel surface with a piece of plastic wrap.
2) Mark the left and right borders of the gel on the plastic wrap with a Texta pen. Expose the X-ray film in a dark room, with the gel placed just above the top layer of the X-ray film. After 1 to 2 min of exposure in the dark, the gel is developed. At this point, the markings on the plastic wrap will appear translucent compared to the gel background on the film, and the successfully labeled RNA will appear as a thicker black band.
3) The markings on the plastic wrap and the film can be used to outline the gel and determine the location of the RNA bands. The resulting gel strip is cut through the plastic wrap and the RNA fragments are recovered as described above.