The scope of our research is to characterize DNA replication of unstable pathogenic repeats in human cells. We hope to answer questions such as, what specific repeats cost replication fork stalling? What unique secondary structures are these repeats forming?
And how do these repeats expand during DNA replication? While we know that many of these structure-forming repeats can impair DNA replication, it's still unclear what exact steps happen next. We're hoping that this protocol will help answer this question and shed light on the mechanisms of how these pathogenic repeats expand.
2D gels provide the unique opportunity to actually visualize the structures in relative populations of molecules that can arise during DNA replication. Within one single experiment, you essentially have the complete map of what replication progression looks like through a specific DNA sequence. To begin, place the reagents required for the preparation of gel electrophoresis on a working platform.
Add Tris-borate-EDTA or TBE to agarose, and heat the solution in a microwave. Then pour the 0.4 to 0.5%agarose into a casting tray to prepare a first-dimension agarose gel, and let it solidify for one hour. After solidification, load the ladder within the first three centimeters relative to the gel's leftmost edge.
Then load the DNA samples digested with restriction enzymes, ensuring a three centimeter distance between each pair. Run the gel in TBE for 19 to 24 hours at 0.85 volts per centimeter to separate the intermediates by size. The following day, remove the gel from the buffer.
Excise the first three centimeters of the gel containing the ladder. Stain the gel segment in TBE containing 0.3 micrograms per milliliter of ethidium bromide for 10 to 15 minutes. Then visualize the ladder using a gel documentation system.
On the agarose gel, add 1.3 centimeters to the estimated location, yielding a value of A.Then subtract 7.5 centimeters from value A, yielding value B.Align the ruler against the first dimension gel, and cut horizontally across at values A and B.Then cut vertically down the three centimeter space reserved for each sample. In a new casting tray, rotate the segments clockwise and place them in the position of the sample wells. Prepare the second dimension agarose gel at a concentration of 1 to 1.3%in TBE with 0.3 micrograms per milliliter of ethidium bromide.
Heat the gel, and upon cooling to approximately 55 degrees Celsius, pour it over the rotated first dimension segments. Allow the gel to solidify for one hour. After solidification, transfer the gel to a chamber containing TBE at 0.3 micrograms per milliliter of ethidium bromide and allow it to equilibrate for 30 minutes.
Cover the gel, and run for nine to 10 hours at 4.23 volts per centimeter at four degrees Celsius. Remove the second dimension gel from the chamber. and depurinate the DNA fragments for 10 minutes in a 0.24 molar hydrochloric acid solution with gentle rocking.
Rinse the gel with deionized water, and soak it in 0.4 molar sodium hydroxide for 10 to 15 minutes. Then fold two long sheets of chromatography paper perpendicular across the glass sheet, extending into the container. To assemble the Southern blot, fill a container with one liter of 0.4 molar sodium hydroxide.
Align a long glass sheet across the container. Wet the top of the paper with sodium hydroxide, and carefully remove any air bubbles under its surface. Soak three sheets of chromatography paper with sodium hydroxide, and place them over the folder paper.
Remove any air bubbles. Flip the second dimension gel upside down, and place it over the chromatography papers. Wet a positively charged nylon membrane with deionized water, and place it over the gel.
Then place three chromatography sheets wet with deionized water over the membrane. Cover any exposed sodium hydroxide in the container with plastic wrap to prevent evaporation. Place a 0.3 to 0.5-meter tall stack of napkins or paper towels over the blot.
Compress the entire blot with a weight to facilitate tight capillary action, and leave it for two days for DNA transfer to the membrane.
