Experiments on the preparation of polyacrylamide gels containing formamide
Experiments on the preparation of polyacrylamide gels containing formamide
A common problem in DNA sequencing is the compression of electrophoretic bands due to the presence of DNA dipolar structures. A simple and straightforward solution is to add formamide to the sequencing gel to reduce the formation of dipolar structures. At G/C levels above 55%, it is almost mandatory to use a gel containing formamide. This experiment is from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by J. Sambrook D.W. Russell.
Operation method
Experiments on the preparation of polyacrylamide gels containing formamide
Materials and Instruments
This methodology includes all items in Option 8 plus: Formamide (100%) Move makings For more product details, please visit Aladdin Scientific website.
This method includes all items in Option 8, plus:
Formamide (100%)
Methods
1. Follow steps 1 to 8 of Option 8 to clean and install the glass plate to make a mold.
2. Cover the working area of the laboratory table with plastic-lined protective paper.
It is almost inevitable that acrylamide solution will drip onto the table when filling sequencing gels, so a large tray can be prepared to collect it.
3. Prepare an appropriate concentration of acrylamide solution in a 250-ml arm conical flask (Table 12-20). This is sufficient to prepare a 40 cm x 40 cm sequencing gel.
IMPORTANT: The gel must be prepared in a single step with no interruptions between the medium and the base.
4. Mix all reagents and heat in a 55°C water bath for 3 min to help dissolve the urea.
The process of dissolving urea is very slow and must rely on external heating.
5. Remove the solution from the water bath and cool for 15 min to room temperature. Stir the mixture continuously. Add water to 100 ml.
6. Place the flask in a vacuum device to evacuate the gas.
This prevents gas bubbles from forming during polymerization, a process that generates heat.
7. Place the solution in a 150 ml glass beaker. Add 3.3 ml of freshly made 1.6% ammonium persulfate and stir the solution gently to mix the reagents. 
8. Add 50ul of TEMED and swirl the container to mix the solution. Dispense the gel directly or draw up 40 ml of the above solution with a 60 ml syringe. Take care that there are no air bubbles.
9. Follow steps 16 to 22 in Option 8.
IMPORTANT: Polymerization often takes 2-3 hours due to the slowing down of the polymerization rate by formamide. 