When cells undergo apoptosis, a series of DNA fragments in a pattern of concatemers of a monomer around 200 bp are formed. This procedure lyses cells, extracts, purifies and tests the DNA fragments using gel electrophoresis.
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When cells undergo apoptosis, the chromatin breaks down between nucleosomes and eventually a series of DNA fragments of an integer multiple of around 200 bp are formed. After these DNA fragments have been extracted, they form a ladder-like appearance on gel by electrophoresis and ethidium bromide or equivalent staining, referred to as DNA Ladders. The DNA fragments are released from the blood and tissue culture cells after the cells are lysed in the lysis / binding solution. The DNA fragments are adsorbed to the silica matrix membrane in the spin column under highly chaotropic salty conditions. Salts, metabolites, proteins and other impurities are then removed through a series of rapid wash and centrifugation steps, and finally DNA Ladder fragments are eluted using the low salt elution buffer.
1. No need to use toxic reagents such as phenol, no need for ethanol precipitation and other steps, either.
2. Unique lysis /binding buffer effectively lyse cells, thus the kit procedure does not require expensive Proteinase K treatment, significantly reducing the cost and procedural time. A single sample can be analyzed within 10 minutes
3. The procedure is easy to use.
Spin Column AC
Materials required and not provided:
Notes on storage:
The product delivers the same performance within 12 months, if stored according to the specified temperature storage conditions.
1. Lysis / Binding Buffer CB may precipitate at low temperature. If precipitates are observed, warm the solution in a 37 ℃ water bath for a few minutes to re-dissolve the precipitates, to restore the clear and transparent appearance. Cool it to room temperature. The solution can then be used.
2. Minimize exposing reagents to air (to avoid evaporation, oxidation, or pH changes). The containers should be promptly closed tightly after each use.
1. All centrifugation steps are done at room temperature using a traditional benchtop centrifuge that can reach 12,000 rpm, such as an Eppendorf 5415C or similar centrifuge.
2. Before starting the experiment, preheat the required water/dry bath to 70 °C. Preheat the elution buffer EB tube in this bath.
3. Lysis / Binding Solution CB contains compounds that may be irritative. Wear latex gloves, goggles and lab coat to avoid contact with skin, eyes and clothing. In case of contact with eyes, or skin, rinse exposed body area with plenty of water or saline wash.
4. Typically, 2 × 106 cultured cells result in a DNA yield of 10-20 μg and 200 μl of human whole blood typically yields 3-6 μg.
5. A typical electrophoresis run calls for a test sample quantity of 2-3μg of purified DNA. If the apoptotic rate is low, it is possible that only the genomic DNA is observed without any DNA ladder. In this case, you can try to increase the sample quantity, both the incoming cells and the quantity to load gel.
Procedural Steps: (Please read the entire instruction before starting the procedure)
Important: Before use for the first time, add the specified amount of absolute ethanol to the wash buffer WB, close the cap and mix thoroughly by inverting the bottle. After adding ethanol, please tick the box on the label with a check mark to avoid adding ethanol subsequently.
1. Take 2 × 106 cells (either suspension cells or adherent tissue culture cells resuspended in 200 μl PBS) or 200 μl
whole blood (containing approximately 2 × 106 cells) and transfer the cells to a 1.5-ml Eppendorf tube. Add 200 μl of lysis / binding solution CB to the tube. Vortex the tube briefly to mix the content thoroughly.
(Optional steps:) If there is too much background RNA, which negatively affects the observation of apoptotic DNA ladder, 20μl RNase A (25mg / ml) solution may be added before adding 200μl lysis / binding buffer CB, mix by shaking and let the tube stand at room temperature for 5-10 minutes .
Note: If necessary, starting volume of cells or whole blood can be increased up to 300μl. If the initial sample volume is increased, the corresponding quantities of reagents in this procedure should also be increased.
2. Incubate the tube(s) at room temperature (15 -20 ℃) for 10 minutes.
3. Add 100 μl of isopropanol and mix well by vortex for 5-10 seconds, at which point a flocculent precipitate may appear.
Note: Sufficient vortex mixing at this step is very important, otherwise, the DNA yield will be reduced. If the sample is viscous, vortex can be extended to a full 15 seconds.
4. Mount a spin column AC onto a collection tube (both are provided). Add the solution (including any precipitate) from last step to the spin column AC. Centrifuge the spin column mounted in collection tube for 30 seconds at 12,000 rpm, and then empty the content in collection tube to a waste container (such as a beaker).
5. Add 700 μl wash buffer WB (check to ensure ethanol has been added!). Centrifuge at 12,000 rpm for 30 seconds and discard the waste.
6. Add 500 μl WB, centrifuge at 12,000 rpm for 30 seconds and discard the waste.
7. With the spin column AC back on the empty collection tube, centrifuged at 12,000 rpm for 2 minutes. This is to remove residual liquid containing leftover ethanol, in order to avoid reduced efficiency of the elution and interference of residual salts in the final DNA solution with downstream reactions.
8a. Remove the spin column AC and place it in a clean centrifuge tube (not provided). Add 100 μl of elution buffer EB in the middle of the adsorption membrane (elution buffer is preheated in a 65-70 ° C water bath). Incubate the column at room temperature 3-5 min. Centrifuge for 1 minute at 12,000 rpm.
8b. The resulting solution is pipetted and added back to the spin column. Let the column stand at room temperature for 2 minutes. Spin the column at 12,000 rpm for 1 minute.
Note: The larger the elution volume is, the higher the elution efficiency is. If the DNA concentration is higher, the elution volume can be reduced appropriately. However, the minimum volume should not be less than 50μl. The smaller the volume, the higher the DNA concentration, though with lower DNA elution efficiency and lower DNA yield.
9. DNA can be used directly or stored at -20 ℃. Ideally the DNA should be analyzed in 2 weeks..
10. Take approximately 2-3 μg of purified DNA for electrophoresis (note that 200 μl of human whole blood typically yields only 3 -6 μg)
Questions and Answers:
Q: There are no DNA Ladder bands, only the genome bands of non-apoptotic cells are observed.
A: Cells are not apoptotic or the rate of apoptotic cells is too low - Recommendations: Increase the concentration of apoptotic agents or prolong the induction of apoptosis
Q: Neither DNA Ladder bands nor genomic bands of non-apoptotic cells were seen
A: Yield of isolation of DNA is too low - Recommendations: Increase the initial quantities of cells, remember to use proportionally increased amounts of reagents. Be sure to perform step 7 to avoid interference of residual salts during DNA elution.
A: Sample itself contains a low amount of DNA (such as human whole blood), thus insufficient quantity of DNA is loaded onto gel. Recommendation: load a larger quantity of purified DNA onto gel, or alternatively test the DNA using an acrylamide gel and silver staining.
Q: DNA bands are diffuse, and no Ladder pattern is observed.
A: This observation is possibly due to non-specific degradation in late-stage Apoptosis. Recommendation: DNA Ladder should be extracted at an earlier stage of apoptosis (preferably, perform a time course of the apoptosis treatment and obtain samples at multiple time points from early, mid, and late-stages).
Q: Background is high, DNA Ladder is weak or not obvious
A: Too much background RNA can obscure the DNA ladder observation – Recommendation: to add DNase free RNase to a final concentration of 2μg / ml into the eluted DNA and incubate the solution at room temperature (15 ° C - 20 ° C) for 20 minutes to degrade RNA. Of note, the normal cells contain more RNA than apoptotic cells. When the proportion of apoptotic cells is too low, background RNA tends to pose a greater problem. Therefore, when a large amount of RNA is observed, it usually suggests that the proportion of apoptotic cells is too low. An optimization of the apoptosis induction conditions is often warranted.