Pouring Polyacrylamide Gels

Last Summer, I worked in the research lab of Dr. Chul Kim where I wrote this protocol for his lab.

Preparation & Usage of Polyacrylamide Gels

Introduction

Polyacrylamide gels are used for the separation of proteins or small nucleic acids. Larger nucleic acids are typically separated using agarose gels. There are two types of polyacrylamide gels used in the laboratory. One type is the denaturing polyacrylamide gels which have urea added. Urea acts as the denaturant and ensures separation of the strands during migration. Typically 20% polyacrylamide gels are used as denaturing gels in our lab. The other types of polyacrylamide gels used are Native gels. These gels do not contain urea and are used to observe the separation of molecules due to conformational differences. Typically 12% polyacrylamide gels are used as native gels in our lab. Since oxygen inhibits the polymerization process, these gels are poured between glass plates.

Materials & Methods

Equipment required:

Measuring cylinder for measuring acrylamide

Micropipettes and micropipette tips

Hot plate with stirrer

Stir bar

Beaker

2 Glass plates (one slightly smaller than the other) and spacers

Gel sealing tape (for small and long gels)

Comb

Binder clips

Casting clamp (for big gels)

Power Supply

Material required (all must be kept refrigerated at 4^oC):

Acrylamide: 12% (without urea) or 20% (containing urea)

10% Ammonium persulfate (APS): catalyst in the polymerization of acrylamide

N,N,N',N'-Tetramethylethylenediamine (TEMED): adjunct catalyst for the polymerization of acrylamide

Quantities required:

Small Gel (~ 20 cm x 20 cm): Acrylamide: 50 ml

APS: 666μl

TEMED: 50μl

Long Gel (~ 35 cm x 20 cm): Acrylamide: 125ml

APS: 1665μl

TEMED: 125μl

Big Gel (~ 41 cm x 33 cm): Acrylamide: 220ml

APS: 2930μl

TEMED: 220μl

Standard Protocol: Acrylamide: 75ml

APS: 1ml

TEMED: 75μl

Method:

1. Clean equipment with hot, distilled and de-ionized water and assemble.
2. Prepare the glass plates:

Place spacers on the longer plate and place the smaller plate on top. If smaller plate has a bevel, point bevel facing inwards on the comb bearing side of the plate. [Make sure that the spacers and the comb are all of the same thickness, 1.6mm.]

Ensure that the spacers and plates are properly aligned and square, the foam pads are pressed tightly to the lower plate and clip the sides with binder clips for small and long gels. Tape the lower end carefully to prevent leakage while pouring for long and big gels, and place a spacer to close the lower border of the small gel. For big gels, close sides using casting clamps.

3. Crumple up paper towels and place beneath corners of plates to catch polyacrylamide leakage.
4. Place an appropriately sized beaker containing a stir bar on a hot plate and set the stir to 8 (high intensity stirring).
5. Take acrylamide from the refrigerator, shake well to mix, and add required quantity to the beaker. Place the acrylamide back before taking the next reagent. [Important: Use the yellow acrylamide measuring cylinder only for the acrylamide]
6. Take APS from the refrigerator, shake well to mix, and add required quantity to the beaker. Replace the APS before taking the next reagent.
7. Take TEMED from the refrigerator, shake well to mix, and add required quantity to the beaker. Replace.
8. Working as quickly as possible, incline the glass plates to a sharp angle and pour the acrylamide mixture continuously into the glass plates along the edge from corner to corner. Take care not to form bubbles while pouring.
9. Once glass plates are filled, place comb while taking care not to push the comb in completely leaving about a mm (pushing the comb in completely causes a suction resulting in the breakage of the gel during removal of the comb). Also make sure that the comb is placed in the center.
10. Place the plates horizontally and let the gel solidify. This typically takes around 20 minutes, minimum (Mol. Clon. Advises one hour). Use the remaining polyacrylamide in the beaker as your guide; if it is solid then your gel is solid. Label plates.
11. These plates can be used immediately or stored at room temperature for 24 hours or at 4°C for 48 hours. To store, do not remove tape or comb, surround top of the gel with paper towels dampened with 0.5X TBE and wrap entire gel with plastic wrap.

Running a Small Gel:

Material Required:

0.5X TBE: 1000 ml

Small electrophoresis chamber, cover and cables

Two binder clips

Electrical unit to set parameters for running the gel

Vaseline

Method:

1. Clean the electrophoresis chamber with hot, distilled and de-ionized water (DI-water).
2. Make up 1 liter of 0.5X TBE by adding 100 ml of 5X TBE and 900 ml of DI-water. Cover with parafilm. Label.
3. Take the poured gel; remove the lower spacer and the comb. Trim any edge, if needed. Wash the wells with DI-water to remove particulate matter. Leave the side spacers in place.
4. With the smaller plate facing inwards, place the plates so they rest snugly against the cushioning. Hold them in place using one binder clip on each side making sure the clips seal the lower edge.
5. Mix the prepared 0.5 X TBE solution. Pour it into the upper chamber to top the gel by about a centimeter. Wait at least 5 minutes to ensure that there are no leakages. If there are any leakages, re-clip the plates and try again. You might also want to change the clips and use tighter binder clips.
6. Once you make sure that there is no leakage, incline the unit and pour 0.5X TBE into the lower chamber. This prevents the formation of air bubbles along the gap created by the removal of the lower spacer. Make sure the lower end of the gel is completely immersed and there are no bubbles below the plate. Bubbles formed on the lower edge can result in improper conductance of charge across the gel.
7. Once the lower chamber is ready, top the upper chamber off so the level is at least two cm above the gel.
8. Map out how you are going to load the wells in advance. When possible, take care not to create a pattern that would look the same. This will allow for the identification of samples even if the gel becomes inverted during the staining process.
9. Add an equal amount of loading buffer with dye to your sample (make sure you pick the right one – native or denaturing, and not the concentrated dye). Heat your sample at 90°C for three minutes.
10. Working as efficiently as possible, rinse out the wells with 0.5X TBE using a syringe. Take care NOT to poke the gel with the needle. This removes any traces of urea from the wells. Leaving urea in the wells will result in improper migration of the sample. As soon as the wells are washed, load 20μl of your sample containing the loading buffer dye into each well. Unless you have very good control over the pipette, push the sample only to the first depression of the pipette. Do not push to the second depression point as you may create a bubble that could flush your sample out of the well.
11. Close the electrophoresis chamber. Label. Plug in the red positive cord to the lower electrode and the black negative cord to the upper electrode. As both RNA and DNA are negatively charged, we run them towards the positive charge.
12. Run the gel at a constant voltage of 300V. Let the gel run for around three hours, until the leading dye is within a cm of the lower border of the gel. Turn off the power and disconnect apparatus.
13. Carefully drain the buffer into the lower chamber and remove the gel plates. Remove the spacers. Separate one glass plate from the gel.
14. Fill a container reserved for staining gels to 1/4^th with toluidine blue stain. Carefully ease the gel into this container.
15. Place the container on a shaker set at low speed. Gentle movement of the stain will allow for better incorporation of the dye into the gel. Let the gel stain for a minimum of one hour or until you can clearly make out that the sample has taken up adequate stain. If staining is not adequate, add a few drops of concentrated stain to the container and let stain longer.
16. Pour the toluidine blue stain carefully into a beaker and pour it back into its original container. Toluidine blue is reused so make sure that it does not get contaminated with polyacrylamide gel pieces. Make sure that you don’t dilute the stain. Hold the gel stable with one hand while pouring the stain to prevent the gel slipping out of the container.
17. Cover the gel with DI-water and place back on the shaker. Keep replacing the water periodically to de-stain efficiently. Failure to change the water may result in the stain settling back on the gel.
18. Once the gel is de-stained, proceed with scanning.

Running a Long Gel:

Materials Required:

Large electrophoresis chamber and cables

Electrical unit to set parameters for running the gel

1 liter 0.5X TBE

Vaseline

Method:

1. Clean the electrophoresis chamber with hot, distilled and de-ionized water (DI-water).
2. Make up 1 liter of 0.5X TBE by adding 100 ml of 5X TBE and 900 ml of DI-water. Cover with parafilm. Label.
3. Take the poured gel; remove the tape and the comb. Trim any edge, if needed. Wash the wells with DI-water to remove particulate matter.
4. With the smaller plate facing inwards, place the plates so they rest snugly on the cushioning. Turn the knobs to hold the plates firmly in place.
5. Make sure the knob connecting the upper and the lower chambers is closed.
6. Mix the prepared 0.5 X TBE solution. Pour it into the upper chamber to top the gel by about two cm. Wait for 5 minutes to ensure that there are no leakages. If there are any leakages, remove the buffer and try to seal the leak with Vaseline.
7. Once you ensure that there are no leakages, pour 0.5X TBE buffer in the lower chamber till the lower end of the gel is completely immersed; about 2 cm above the gel.
8. Map out how you are going to load the wells in advance. When possible, take care not to create a pattern that would look the same. This will allow for the identification of samples even if the gel becomes inverted during the staining process.
9. Add an equal amount of loading buffer to your sample (make sure you pick the right one – native or denaturing, and not the concentrated dye). Heat your sample at 90°C for three minutes.
10. Working as efficiently as possible, rinse out the well with 0.5X TBE using a syringe. This removes any sediments of urea from the wells. Leaving urea in the wells will result in improper migration of the sample. As soon as the wells are washed, load the sample into your well.
11. Make sure that you do not poke the gel with the loading tip. If the tip gets clogged, remove, trim the end using a sharp scissors and continue loading.
12. Plug in the red positive cord to the lower electrode and the black negative cord to the upper electrode. As both RNA and DNA are negatively charged, we run them towards the positive charge. Label the unit.
13. Run the gel at a constant voltage of 300V. Let the gel run for around seven hours, until the leading dye is within a cm of the lower border of the gel. Turn off the power and disconnect apparatus.
14. Open the knob connecting the upper and lower chambers. Carefully drain the buffer into the lower chamber and remove the gel plates. Remove the spacers. Separate one glass plate from the gel.
15. Fill a container reserved for staining gels to 1/4^th with toluidine blue stain. Carefully ease the gel into this container.
16. Place the container on a shaker set at low speed. Gentle movement of the stain will allow for better incorporation of the dye into the gel. Let the gel stain for a minimum of one hour or until you can clearly make out that the sample has taken up adequate stain. If staining is not adequate, add a few drops of concentrated stain to the container and let stain longer.
17. Pour the toluidine blue stain carefully into a beaker and pour it back into its original container. Toluidine blue is reused so make sure that it does not get contaminated with polyacrylamide gel pieces. Make sure that you don’t dilute the stain. Hold the gel stable with one hand while pouring the stain to prevent the gel slipping out of the container.
18. Cover the gel with DI-water and place back on the shaker. Keep replacing the water periodically to de-stain efficiently. Failure to change the water may result in the stain settling back on the gel.
19. Once the gel is de-stained, proceed with scanning.

Running a Big Gel:

Materials required:

Large electrophoresis chamber and cables

Electrical unit to set parameters for running the gel

1 liter 0.5X TBE

Vaseline

Method:

Clean the electrophoresis chamber with hot, distilled and de-ionized water (DI-water).

Make up 1 liter of 0.5X TBE by adding 100 ml of 5X TBE and 900 ml of DI-water. Cover with parafilm. Label.

Take the poured gel; remove the tape and the comb. Trim any edge, if needed. Wash the wells with DI-water to remove particulate matter.

With the smaller plate facing inwards, place the plates so they rest snugly on the cushioning. Turn the knobs to hold the plates firmly in place.

Make sure the knob connecting the upper and the lower chambers is closed.

Mix the prepared 0.5 X TBE solution. Pour it into the upper chamber to top the gel by about two cm. Wait for 5 minutes to ensure that there are no leakages. If there are any leakages, remove the buffer and try to seal the leak with Vaseline.

Once you ensure that there are no leakages, pour 0.5X TBE buffer in the lower chamber till the lower end of the gel is completely immersed; about 2 cm above the gel.

Map out how you are going to load the wells in advance. When possible, take care not to create a pattern that would look the same. This will allow for the identification of samples even if the gel becomes inverted during the staining process.

Add an equal amount of loading buffer to your sample (make sure you pick the right one – native or denaturing, and not the concentrated dye). Heat your sample at 90°C for three minutes.

Working as efficiently as possible, rinse out the well with 0.5X TBE using a syringe. This removes any sediments of urea from the wells. Leaving urea in the wells will result in improper migration of the sample. As soon as the wells are washed, load the sample into your well. The large well can contain a maximum of 2 ml of sample + dye. Use a 300μl gel loading tip and load sample in increments in a layering motion. Make sure that you do not poke the gel with the loading tip. If the tip gets clogged, remove, trim the end using a sharp scissors and continue loading.

Plug in the red positive cord to the lower electrode and the black negative cord to the upper electrode. As both RNA and DNA are negatively charged, we run them towards the positive charge. Label the unit.

Run the gel at a constant power of 45 Watts per each big gel for approximately 6500 Volt hours. With this setting, the gel is usually allowed to run overnight.

Turn off the power and disconnect apparatus.

Open the knob connecting the upper and lower chambers. Carefully drain the buffer into the lower chamber and remove the gel plates. Remove the spacers. Separate one glass plate from the gel. Take a large sheet of plastic wrap and carefully ease the gel onto the wrap.

Place the wrap on reflective squares which fluoresce when irradiated with short wavelength UV rays.

Identify the sample and cut the gel into 0.5cm pieces using a clean scalpel. Transfer the pieces into a clean falcon tube using a clean forceps. Label and store at -20°C until ready to extract the sample using an ELUTRAP.

Conclusion

1. Acrylamide is a neurotoxin. Hence, all work should be conducted wearing gloves. Special care should be taken to ensure that the appropriate measuring cylinder alone is used. Acrylamide [liquid] should never be disposed down the sink.
2. Polyacrylamide or the polymerized acrylamide is not a toxin, and can be discarded in the trash.
3. If unsure about the procedure, polymerization time can be delayed by placing the acrylamide, APS, TEMED mixture on ice.
4. After running the gel, TBE buffer used can be discarded down the sink.
5. Toluidine blue stain is reused. Place back into original container after use. DI-water containing stain can be discarded down the sink.

References

“Molecular Cloning: A Laboratory Manual”, Second Ed., by J Sambrook, E.F. Fritsch and T Maniatis (1989) 12.74 – 12.80

http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/principles.html

Introduction

Like all good beginnings, I would like to introduce myself and my work. I am a Master's student in Cell and Molecular Biology, and am also currently doing a certificate program in Biotechnology. My undergraduate degree was in Dentistry. After completion of my Dental degree from SRMC & RI, I came to California, where I completed one year of Post-baccalaureate studies in Cell and Molecular Biology. On completion of the above, I was fortunate enough to be accepted into the Master's program at Cal State, East Bay, which brings us up to date.

My Mentor: Every person has an idol, a guru, and mine is Professor Chris Baysdorfer. An untiring perfectionist, his ability to go from class to class and from topic to topic leaves all of us amazed.

In the subsequent posts, I will try to put my experiences, projects and reviews online. Since I am currently taking the following four courses, my posts in the next few months will cover these topics mostly.

Courses lined up:
PCR, DNA seqencing and Fragment analysis
Cell and Molecular Biology
Bioinformatics
Molecular Biology Seminar