6% Polyacrylamide Gel (TBE system)

Md Jubayer Alam

Apr 13, 2026

6% Polyacrylamide Gel (TBE system)

DOI

https://dx.doi.org/10.17504/protocols.io.e6nvww7o9vmk/v1

Md Jubayer Alam¹

¹Lakehead University

Md Jubayer Alam

Md Jubayer Alam

Lakehead University

DOI: https://dx.doi.org/10.17504/protocols.io.e6nvww7o9vmk/v1

Protocol Citation: Md Jubayer Alam 2026. 6% Polyacrylamide Gel (TBE system). protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvww7o9vmk/v1

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: April 09, 2026

Last Modified: April 13, 2026

Protocol Integer ID: 314786

Keywords: nucleic acid electrophoresis under tbe buffer condition, nucleic acid electrophoresi, gel polymerisation, ready procedure suitable for routine dna analysis, primary components for gel polymerisation, routine dna analysis, ml gel solution, reliable gel performance, acrylamide, polymerisation, dna, ethidium bromide, incomplete polymerisation, electrophoretic separation

Abstract

This protocol describes the preparation and use of a 6% polyacrylamide gel for
nucleic acid electrophoresis under TBE buffer conditions. A 30% acrylamide/bis-acrylamide
stock and a freshly prepared 10% ammonium persulfate (APS) solution serve as
the primary components for gel polymerisation, initiated by TEMED. The method
outlines precise reagent ratios for generating a 20 mL gel solution, followed
by casting, polymerisation, and electrophoretic separation at constant voltage.
Post-run visualisation is achieved through ethidium bromide staining and UV
illumination. A troubleshooting guide addressing common issues, including
incomplete polymerisation, smiling bands, overheating, and distorted wells,
supports reliable gel performance. This workflow provides a reproducible,
laboratory-ready procedure suitable for routine DNA analysis and teaching
applications.

Guidelines

1. Reagents required**
   - 30% Polyacrylamide solution
   - 10% APS (Ammoniumperoxodisulfate)
   - Distilled water
   - 5X TBE (Tris-borate-EDTA) buffer
   - 1X TBE (Tris-borate-EDTA) buffer
   - TEMED (N, N, N, N- Tetramethylethylenediamine)

2. 30% Polyacrylamide Solution Preparation (Stock solution)**
- 2.1 30% Polyacrylamide/Bis Solution (Stock)**
     1. Dissolve 29g of acrylamide and 1g of bis-acrylamide in about 60mL of distilled water.
     2. Add water until the total volume reaches 100mL.
     
Note: Store at 4 °C (protect from light)_
   
- 2.2 10% APS preparation**
     1. Dissolve 0.033g APS in 330 µl of distilled water
     
Note: Store at 4 °C (prepare fresh weekly). 

3. Preparation of 6% Polyacrylamide Gel (20 mL Total Volume)**
- 3.1 Gel Solution Preparation**
     1. Take 12 mL of distilled water in a clean glass beaker.
     2. Add 4ml 30% Polyacrylamide solution to it.
     3. Add 4 ml 5X TBE buffer.
     4. Stir gently until the mixture is homogeneous.
   
- 3.2 Initiation of Polymerisation**
     5. Add 330 µl of 10% APS while stirring
     6. At last, add 16.7 µl TEMED and mix quickly.
     
Note: APS and TEMED are highly reactive. Therefore, we should always add them last, just seconds before pouring the liquid into the glass plates (keep stirring). 

4. Casting the Gel**
   1. Immediately pour the gel solution between gel plates (polymerisation begins within seconds)
   2. Insert the comb carefully to form wells.
   3. Allow the gel to polymerise (approximately 30-35 min) at room temperature.
   4. Remove the comb once the gel is fully solidified.
   5. Rinse wells gently with buffer using a pipette to remove trapped air bubbles.

5. Electrophoresis**
   1. Pre-run the gel (without samples) in 1X TBE at 120 V for 5 minutes.
   2. Load samples into wells.
   3. Run the gel at 120 V until the dye front reaches the bottom or runs off the gel.

6. Ethidium Bromide Staining**
   1. Stain in a 0.5% ethidium bromide solution for 15 min
   2. Rinse briefly with distilled water to enhance contrast
   3. Visualise the gel under a UV transilluminator.

**Troubleshooting Guide for Polyacrylamide Gels**

1. Smiling Bands (Curved Upward in the Middle)**
   
❖ Possible Causes_
   - Overheating during electrophoresis.
   - Uneven heat dissipation (thick gel, poor buffer circulation).
   - Running voltage too high.
   - Buffer too old or too concentrated.
   
➢ Fixes_
   - Reduce voltage (e.g., from 120 volts to 80-100 volts).
   - Ensure fresh 1X TBE is used.
   - Ensure gel plates are fully submerged and parallel.
   - Use thinner gels for high-voltage runs.

2. Incomplete or Failed Polymerisation**
   
❖ Possible Causes_
   - Old APS (decomposed).
   - TEMED too old or oxidised.
   - Acrylamide stock degraded (especially if stored warm or exposed to light).
   - Incorrect APS/TEMED volumes.
   - Contamination with SDS, reducing agents.
   - Oxygen exposure (oxygen inhibits free-radical polymerisation).
   
➢ Fixes_
   - Prepare fresh 10% APS (best within 1 week)
   - Use fresh TEMED
   - Mix APS and TEMED immediately before pouring
   - Ensure glass plates are clean and dry
   - Avoid stirring too vigorously (introduces oxygen)

3. Overheating During Electrophoresis**
   
❖ Possible Causes_
   - Voltage is too high
   - Buffer too concentrated (e.g., accidentally using 5X TBE buffer instead of 1X TBE buffer)
   - Running buffer volume too low
   - Poor heat dissipation (thick gel, warm room)
   
➢ Fixes_
   - Confirm buffer is 1X TBE
   - Reduce voltage
   - Increase buffer volume in the tank

4. Distorted or Irregular Bands**
   
❖ Possible Causes_
   - Uneven polymerisation
   - Air bubbles in wells
   - Damaged or irregular wells
   
➢ Fixes_
   - Rinse wells with water before loading
   - Load samples slowly to avoid tearing wells
   - Spin samples briefly to remove bubbles
   - Ensure consistent loading volumes

5. Bands Running Too Slowly**
   
❖ Possible Causes_
   - Gel percentage too high
   - Running buffer too concentrated
   - Voltage too low
   - Gel polymerised too long before use (drying or stiffening)
   
➢ Fixes_
   - Confirm correct gel percentage (6% here)
   - Use fresh 1X TBE
   - Increase voltage moderately

6. Bands Running Too Fast**
   
❖ Possible Causes_
   - Gel percentage too low
   - Running buffer too dilute
   - Voltage too high
   - Overheating accelerates migration
   
➢ Fixes_
   - Confirm correct gel percentage
   - Replace buffer with fresh 1X TBE
   - Reduce voltage

7. Smearing**
   
❖ Possible Causes_
   - Overloaded sample
   - Degraded sample
   - Incomplete polymerisation of the sample
   - Running buffer exhausted
   
➢ Fixes_
   - Reduce sample volume
   - Purify sample
   - Prepare fresh gel
   - Replace running buffer
   - Ensure complete polymerisation of the sample before loading

8. Wells Collapse or Tear**
   
❖ Possible Causes_
   - Gel not fully polymerised
   - Comb removed too early or too forcefully
   - Gel too thin
   - APS or TEMED imbalance
   
➢ Fixes_
   - Allow full 30–35 minutes for polymerisation
   - Remove comb slowly, straight upward
   - Use correct spacer thickness
   - Verify APS and TEMED volumes

9. Weak or Faint Bands After Ethidium Bromide Staining**
   
❖ Possible Causes_
   - Low DNA concentration
   - Short staining time
   - Old ethidium bromide
   - Over-washing
   - UV exposure too brief
   
➢ Fixes_
   - Increase staining time (15–20 min)
   - Use fresh Ethidium Bromide
   - Reduce washing time
   - Increase UV exposure duration (but avoid overexposure)

Materials

30% Polyacrylamide solution
10% APS (Ammoniumperoxodisulfate)
Distilled water
5X TBE (Tris-borate-EDTA) buffer
1X TBE (Tris-borate-EDTA) buffer
TEMED (N, N, N, N- Tetramethylethylenediamine)

Safety warnings

APS and TEMED are highly reactive. Therefore, we should always add them last, just seconds before pouring the liquid into the glass plates (keep stirring).

30% Polyacrylamide Solution Preparation (Stock solution)

1.1. Dissolve 29 g of acrylamide and 1 g of bis-acrylamide in about 60 mL of distilled water.
1.2. Add water until the total volume reaches 100 mL.
Note: Store at 4°C (protect from light).

10% APS Preparation (For 20 mL solution)

Dissolve 0.033g APS in 330 µl of distilled water
Note: Store at 4°C (prepare fresh weekly).

Preparation of 6% Polyacrylamide Gel (Around 20 mL Total Volume)

1. Take 12 mL of distilled water in a clean glass beaker.
2. Add 4 mL 30% Polpolyacrylamidelution to it.
3. Add 4 mL of 5X TBE buffer.
2.  Stir gently until the mixture is homogeneous.

Initiation of Polymerization

4.1. Add 330 µl of 10% APS while stirring
4.2. At last, add 16.7 µl TEMED and mix quickly.
Note: APS and TEMED are highly reactive. Therefore, we should always add them last, just seconds before pouring the liquid into the glass plates (keep stirring).

Casting the Gel

35m

1. Immediately pour the gel solution between gel plates (polymerisation begins within seconds)
2. Insert the comb carefully to form wells.
3. Allow the gel to polymerise (approximately 30-35 min) at room temperature.
4. Remove the comb once the gel is fully solidified.
5. Rinse wells gently with buffer using a pipette to remove trapped air bubbles.

Polyacrylamide Gel Electrophoresis

1. Pre-run the gel (without samples) in 1X TBE at 120 V for 5 minutes.
2. Load samples into wells.
3. Run the gel at 120 V until the dye front reaches the bottom or runs off the gel (around 50-55 min).
 

Ethidium Bromide Staining

15m

1. Stain in a 0.5% ethidium bromide solution for 15 min.
2. Rinse briefly with distilled water to enhance contrast.
3. Visualise the gel under a UV transilluminator.