This article details a method for analyzing charged and uncharged E. coli tRNAs using denaturing polyacrylamide gel electrophoresis and subsequent hybridization techniques. The study aims to differentiate between charged and uncharged tRNAs based on their migration patterns during electrophoresis.
Begin with E. coli tRNAs, containing both charged forms with an attached amino acid and uncharged forms without the amino acid.
Include uncharged tRNAs as a control.
Mix the samples with a loading buffer containing tracking dyes and denaturing agents.
Load the samples onto a denaturing polyacrylamide gel and perform electrophoresis.
Denaturing conditions linearize the tRNAs.
Charged tRNAs migrate more slowly than uncharged ones due to the attached amino acid.
Excise the gel region containing the tRNAs and transfer them to a positively charged nylon membrane via electroblotting.
Immobilize the tRNAs on the membrane using UV crosslinking.
Insert the membrane into a tube, add hybridization solution, and incubate to reduce non-specific probe binding.
Add radiolabeled probes targeting the tRNAs.
Wash off unbound probes.
Using a radioimager, detect the slower-migrating band indicating charged tRNAs and the faster band matching the control representing uncharged tRNAs.
To start electrophoresis, mix four microliters of each sample with six microliters of loading buffer.
Load the samples including controls onto a 0.4 millimeter thick 6.5 percent polyacrylamide gel containing eight molar urea and 0.1 molar sodium acetate buffer. Then, separate the RNA by running the electrophoresis at 10 volts per centimeter of gel at four degree celsius for approximately 20 hours until the bromophenol blue dye reaches the bottom of the gel. Carefully separate the two glass plates so that the gel remains on one of them.
Use the gel area from the xylene cyanol dye and 20 centimeters down towards the bromophenol dye for blotting. Cut a thin piece of filter paper to the size of the desired blotting area. Place the filter paper on top of the part of the gel that will be used for blotting.
Cut off and discard the parts of the gel not covered with filter paper. Then use the filter paper to carefully lift the gel away from the glass plate. Place a positively charged nylon membrane on top of the gel, and electroblot it in transfer buffer at 20 volts for 90 minutes.
Finish by crosslinking the RNA to the membrane using UV light. Next, place the crosslinked membrane in a hybridization tube and add six milliliters of hybridization solution. Rotate the tube at 42 degree celsius for one hour to pre-hybridize the membrane.
Then, add 30 picomolar radioactive oligo DNA probe, five prime end labeled with 32P. Rotate to incubate the membrane with the probe at 42 degree Celsius overnight. After incubation, use a disposable 10 milliliter pipette to remove the probe.
Next, quickly wash the membrane twice in the hybridization tube, in 50 milliliters of washing solution at room temperature. Move the membrane to a flat plastic container, cover it with the washing solution and close the lid. Incubate the membrane on a shaker at room temperature for 30 minutes.
Change the solution and continue washing until achieving a satisfying signal to noise ratio. Use a Geiger-Müller tube to obtain the signal to noise ratio, by comparing the radiation from the area where the probe has hybridized to tRNA, to the radiation from an empty area. After that, wrap the membrane tightly in plastic wrap and seal it airtight by welding.
Place this membrane on a phosphorimaging screen. After adequate exposure time, scan the screen using a laser scanner and save the file in dot gel format.
繁體中文
