In general, this type of gel is required to separate single stranded DNA or RNA fragments such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions. The separation range is dependent on the polyamide concentration, uria in combination with heat denas to sample and single stranded molecules migrate within the gel matrix according to their molecule weight. This video presents the pouring, running, and processing of a typical gel.
Hello, my name is Haki. I'm with Nang Technological University. In this demonstration, I'm going to show you how to perform A Denaturing poly gel electrophoresis.
Use clean glass plates and assemble the gel Sandwich according to manufacturer's description. Fix the gel in the gel casting standard chamber. Ize the glass plates of large gels if the gel tends to stick to the glass plates.
Choose a gel size and thickness according to your separation requirements. Prepare the gel solution. According to current protocols in molecular biology.
Choose a concentration of polyamide that suits your separation requirements. Best higher percentage of acrylamide will resolve lower molecular weight fragments. Use Alta pure UIA and mixed with the desired amount of poly acrylamide in a beaker or bottle.
Add 10 XTBE buffer to the solution and fill up to the required volume with water. Heat the solution for 20 seconds in the microwave and mix it gently until all UIA is dissolved. You can store the solution if necessary for larger gel volumes.
Repeat the microwave step until the solution is hand warm. Avoid heating the solution too much. Do not overheat because uria will start to degrade after 60 degrees.
Centigrade can be detected by the smell of ammonia. When the solution is clear and has cool to about room temperature, add a PS and tamid to the gel solution and gently mix it. If the gel solution is too warm, the gel will polymerize inside the pipette.
Pour the gel using a serological pipette and then pipette aid between the two glass plates. Avoid introducing air bubbles. Insert tecom carefully.
Try not to trap any air bubbles and let the gel polymerize For 30 to 60 minutes. Dismount the gel from the Casting stand and assemble it to the upper buffer chamber. According to manufacturer's protocol, put the gel into the lower buffer chamber and fill the bottom reservoir with gel running buffer, which is normally 0.5 to one XTBE.
The same concentration as for the gel, fill the upper buffer chamber with gel running buffer up to the edge of the glass plate. Before you can load your samples, you have to pre-run the gel for at least 30 minutes to heat up the gel and remove remaining ure from the gel. The optimal temperature should be between 45 to 55 degrees centigrade, like for the actual gel run.
Avoid temperatures higher than 60 degrees as bands could smear or the glass plates could crack carefully. Remove the calm without disturbing the edges and rinse the wells through it to remove ure that has leaked into them. Use gel loading tips and pipe it up and down several times.
For each, well attach the lid of the gel system and plug in the cables to a high voltage power supply. Choose constant watts for the pre-run, about 15 to 20 watts per gel. When the power supply switched on, air bubbles indicate good conductivity.
In the meantime, prepare your samples, therefore at the appropriate Amount of two x gel loading to your sample. When the pre-run is finished, remove the lid and Wash the pocket throughly as described before, it is very important to carefully rinse the gel pockets. To remove uria residues as uria will form a dense layer that hinders the sample from entering the gel.
Right before you. Apply your sample heating nature, your sample by incubating it to at least 70 degrees centigrades for several minutes. Apply the samples carefully from the bottom of the pocket.
For some applications, the dye interferes with your sample and can cause diffuse bands, or the dye shows the fluorescent signal. Then it is useful to remove one dye or all dyes and load the standard dye containing buffer. In an empty well a size standard, the amount of sample you should load for sharp bands is ideally between one to five microliters, but you can load higher volumes up to 15 microliters as well.
Less sample volume results in nicer bands. Avoid introducing air bubbles and let the sample sink into the pocket slowly that a unified layer is formed. If you use a fluorescent label, pack the gel system into aluminum fall to prevent exposure to light, which could cause photobleaching of your sample.
Assemble the lid and switch on the power supply. Run the gel until the DIA front reached the lower end of the gel. The run duration is dependent on the percentage of acrylamide.
You choose iion strength of your buffer gel thickness and length. A run can last between one to several hours. Observe the migration of the marketized to determine the electro freeze duration.
Use constant watts and check the temperature on the gel. A gel thermometer can help to monitor the right temperature during the run or check the manual of the gel Apparatus when the dron has reached the end. Switch off the power supply and remove the lid of the gel apparatus.
Next, put the gel sandwich out of the lower buffer chamber. Pour away the buffer from the upper buffer chamber. Fill a dish with gel fixation solution.
This solution should have the same concentration of TB and between five to 10%methanol and aesthetic acid. Remove the gel sandwich from the apparatus and loosen the clamps. Pull away the spacers and carefully disassemble the glass plates using a spatula If necessary, cut the weighter up a well containing gel part or the lower empty gel part and discard the needless gel pieces like dye containing lanes.
For fluorescent samples, carefully detach the gel from the glass plate and transfer it into a dish filled with gel fixation solution. Leave the gel into the solution for five to 10 minutes and change the buffer twice. Gently aate the dish to wash out the ure from the gel or put the gel in staining solution depending on your application.
The gel quality is also dependent on the gel thickness. Thinner gels like 0.75 millimeter show better results than 1.5 millimeter thick gels, but are more difficult to handle. The sharpness of the bands depends on several factors.
Low voltage at the beginning of the run can help to get nicer bands as well as the sample enters gel front smoothly, and this avoids that the gel pockets collapse due to high voltage. A short, low percentage acrylamide stacking gel can have the same band sharpening effect, including 10%Glycerol also helps during the gel loading and the sample sinks into the well. More easily apply loading buffer to empty pockets to maintain equal conditions during the run.
When your gel is in fixation solution, you can dry your gel using a vacuum gel dryer for further imaging or scan your gel directly depending on label thigh or stain you are using. This U page is useful to analyze or isolate single stranded DNA or RNA samples. Gels can be stored at room temperature.
Therefore, wrap the gel in paper towel and soak it with water or running buffer. Put the wrapped and wet gel into a seal level plastic bag and use it within 48 hours. An often observed problem is gel smiling.
This is due to uneven heat distribution through the gel. If the gel is not surrounded by buffer, attaching of a metal plate can increase the quality of the gel. Hi, I'm Hi Kasam.
I'm with N Yang Technological University. I showed you how to perform a denaturing Polymeal pheresis. See you next time.
