Cellular DNA Damage Detection
Cellular DNA Damage Detection
Principle
When the cellular DNA chain is broken, the superhelical structure of DNA is damaged, in neutral conditions, DNA fragments can enter the gel to migrate, while under the action of alkali treatment and alkaline electrolyte, DNA is de-helicalized, and the damaged DNA broken strands and fragments are released, and because of the small molecular weight of these DNAs, they leave the nucleus DNA to move toward the anode during electrophoresis to form a comet-like trailing image, while the undamaged DNA part stays in the original position to form a round fluorescent cluster after staining. Because of the small molecular weight of these DNAs, they will leave the nuclear DNA and move toward the anode during electrophoresis, forming a comet-like trailing image, while the undamaged DNAs will stay in the original position and become a round fluorescent cluster after staining.
Appliance
Evaluation of DNA damage in individual cells (biological response to various factors, tumor research treatment prediction).
Operation method
Single-cell gel electrophoresis experiment
Principle
When the cellular DNA chain is broken, the superhelical structure of DNA is damaged, in neutral conditions, DNA fragments can enter the gel to migrate, while under the action of alkali treatment and alkaline electrolyte, DNA is de-helicalized, and the damaged DNA broken strands and fragments are released, and because of the small molecular weight of these DNAs, they leave the nucleus DNA to move toward the anode during electrophoresis to form a comet-like trailing image, while the undamaged DNA part stays in the original position to form a round fluorescent cluster after staining. Because of the small molecular weight of these DNAs, they will leave the nuclear DNA and move toward the anode during electrophoresis, forming a comet-like trailing image, while the undamaged DNAs will stay in the original position and become a round fluorescent cluster after staining.
Materials and Instruments
Reagent] Low melting point agarose, normal melting point agarose Move 1、Preparation of single cell suspension Cells were digested into centrifuge tubes with trypsin and counted, and the cell density was adjusted to 105~106 cells/ml with PBS.
PBS, TBE (5X), ethidium bromide (EB)
Tris-HCl solution, RIPA cell lysate
Slides, coverslips
[Equipment] Fluorescence microscope, nucleic acid electrophoresis apparatus
The first layer was made by dropping 80 μl of 0.5% normal melting point agarose onto a preheated slide, quickly covered with a clean coverslip and placed at 4 ℃ for 10 min to solidify.
The second layer was a mixture of low melting point agarose and cells, 10 μl of PBS containing 10,000 cells and 75 μl of 0.5% low melting point agarose were mixed well at 37 ℃, then the coverslip was gently removed, and a drop of cell-containing low melting point agarose was dropped onto the first layer of the gel plate, which was then covered with a clean coverslip immediately and placed at 4 ℃ for 10 min to solidify.
The third layer of gel was prepared in the same way as the first layer of gel and covered with a coverslip.
3、LysisRemove the coverslip and immerse the slide in freshly prepared RIPA cell lysis solution for at least 2.5 h (try to make the lysis time the same for each slide). The purpose of this step is that the lysate consists of a descaling agent and a high salt solution, which removes the lysed cell and nuclear membranes, removes proteins, RNA, etc., and leaves only the nucleoskeleton.
4. Unspinning and electrophoresisAfter cell lysis, take out the slide and rinse it with PBS for 2 times, then put the slide in a horizontal electrophoresis tank, pour 1XTBE electrophoresis buffer, cover the surface of the gel with 0.25 cm, and despin with alkaline solution for 20 min, and then electrophoresis in the refrigerator at 4 ℃ for 25~30 min at a voltage of 25 V. The electrophoresis was carried out in the refrigerator at 4 ℃ for 25~30 min.
5. StainingRemove the slides, absorb the electrophoresis buffer with filter paper, neutralize with Tris-HCl (pH 7.5) for 15 min, then add 50 μl of 30 μg/mL ethidium bromide (EB) solution onto each slide, avoid light, cover the coverslip, and then stain the slides avoiding light for 20 min, and then observe.
6. Microscopic nuclear image analysisAfter EB staining, the samples were observed under a fluorescence microscope as soon as possible and electrophoretic images were taken.
Caveat
1, Incomplete cell digestion, did not get a single cell, multiple cells occurred adhesion.
2， The shape of the tail is incomplete or twisted.
3， lysate treatment is the key to this experiment, lysate should be clarified and completely dissolved, lysis is incomplete to extend the lysis time or consider the use of strong lysate.
4、Ethidium bromide is a highly toxic substance, need to be well protected, can consider using other nucleic acid dyes instead. Common Problems Question 1: The cell digestion is not complete, individual cells are not obtained and multiple cells become adherent. A: The cell digestion process is carried out in an incubator for digestion and can be done by gently blowing with a pipette gun. Problem 2: The tail is incomplete or twisted. A: The glue making process is meticulous and careful, do not produce air bubbles, the glue surface is flat. Strip the glue carefully and meticulously. Keep the electrophoresis at low and smooth voltage, electrophoresis in 4 ℃ refrigerator. For more product details, please visit Aladdin Scientific website.