Unlike other kits from our peers, this Apoptotic DNA Ladder HS (High Sensitivity/Selectivity) Kit selectively extracts apoptotic DNA ladder, leaving behind intact genomic DNA of normal tissues and cells, thus significantly improving the detection sensitivity of DNA ladders. The HS kit is convenient-the procedure is carried out in an Epperforf tube, without organic extraction and takes 2.5 hours.
This product is no longer in stock
A morphologically significant feature of apoptosis or programmed cell death is that the chromosomal DNA is regularly cleaved in nucleosomes (185 bp) to a length of approximately n × 185 bp (n = 1, 2, 3, 4 ... ) Of DNA fragments. Presentation of ladder-like DNA ladder by agarose gel electrophoresis, is thus a hallmark of apoptotic cells. Unlike other kits from our peers, this Apoptotic DNA Ladder HS (High Sensitivity/Selectivity) Kit selectively separates and extracts apoptotic DNA ladder from intact genomic DNA of normal tissues and cells. By selectively removing genomic DNA from apoptotic DNA ladder, the procedure minimizes the obscuring effect of genomic DNA on apoptotic DNA ladders, thus significantly improving the detection sensitivity of DNA ladders. The HS kit is convenient and fast-the reaction can be carried out in a microcentrifuge tube, without organic extraction and takes 2.5 hours to complete. The high detection sensitivity means that DNA ladders can be observed from only about 2,000 apoptotic cells. We recommend initial cell number of 5 to 10 × 105 cells, though the number of cells can vary from 1 × 105 to 5 × 106, provided that the total cells contain at least about 1-2 × 104 apoptotic cells. More than 2 × 104 apoptotic cells can usually produce a very clear apoptotic DNA ladders. This kit can also be used to extract apoptotic DNA ladder from tissues. However, compared with cultured cells, apoptotic cells in the whole animal tissue are influenced by multiple factors, such as time, tissue morphology/location and extent of apoptosis, thus often varied experimental results are observed with tissues. Nonetheless, as long as the tissue undergoes apoptosis, an experienced user may be able to use this kit to extract apoptotic DNA ladders from the tissue (see Note 4).
Materials required and not provided:
6x gel loading buffer
DNAase free water
Precautions on reconstitution and storage:
In order to provide convenience in transport and extend stability, Enzyme B is provided as a lyophilized powder. After receiving it, the user is to add 500 μl (25 uses) or 1 ml sterilized water (50 uses), depending on the kit size, to resuspend the enzyme. Store the resuspended enzyme at -20 ºC. Avoid repeated freezing and thawing of Enzyme A and Enzyme B solutions to ensure the full activity. If you want to use the kit multiple times, it is best to aliquot the enzymes and store the aliquots at -20 º C.
1. Overstaining of gel by ethidium bromide will reduce the detection sensitivity of DNA bands. To mitigate this problem, the gel can be washed with water for 10 to 30 minutes to dampen the staining. Under-staining can be mitigated with an added ethidium bromide staining step. The more sensitive DNA stain SYBR Green can be used instead of ethidium bromide to increase detection sensitivity of this procedure. Acrylamide DNA gel electrophoresis and DNA silver staining can be used instead of agarose gel and fluorescent staining to increase the detection sensitivity of DNA ladders.
2. After induction of the cells, apoptosis may be most evident only at a certain point in time or at a certain intensity of intervention. A pretest is therefore needed to determine the optimal time or intensity of intervention to capture the conditions under which apoptosis is most obvious. This pretest can be aided by an apoptotic bodies / Hoeschst staining kit to rapidly stain and observe apoptotic bodies.
3. Recommended starting cell number is 5 - 10 × 105, but the number of cells used can vary between 1 ×105 and 5 ×106. The rule of thumb is that the total cells should contain at least about 1-2 × 104 apoptotic cells. More than 2 × 104 apoptotic cells can usually result in a very clear apoptotic DNA ladder. One well in a six-well plate corresponds to one 35 mm Petri dish. When full, up to 1 to 10 × 10 5 cells can be obtained. If the rate of apoptosis is 10%, about 1 to 10 × 104 apoptotic cells can be obtained after treatment, which should be enough to obtain a clear apoptotic DNA ladder. Conversely, if a clear apoptotic DNA ladder cannot be obtained from > 3 × 106 cells, less than 1% of apoptotic cells is indicated. In this case, increasing the number of cells won’t offset the low percentage of apoptosis.
4. Extraction of apoptosis DNA ladder from the tissue block: Take 10 to 20 mg tissue block into a glass homogenizer, add 100 to 200 µl Extraction buffer, manually homogenized up and down 15 to 20 times. Transfer the homogenate to a microcentrifuge tube, place the tube on ice for 5 to 10 min.
Vortex the tube for 10 seconds. Centrifuge the tube at 4500 rpm for 10 minutes and transfer supernatant to a new 1.5 ml centrifuge tube and perform the extraction step 3 and subsequent steps below.
Another method is to mince the 30- 50 mg tissue block into small pieces, homogenize the tissue in PBS into a cell suspension. Centrifuge the tube to collect cells, perform step 2 and subsequent steps of the extraction procedure below.
5. In order to ensure good resolution of apoptotic DNA ladders, use high quality agarose, and thinner, narrower sample combs to make thinner agarose gels (about 2 to 4 mm thick). Slow electrophoresis at lower voltages will significantly increase Apoptotic DNA band resolution. On the other hand, also ensure that the electrophoresis does not go on for too long a period (30-60 min is ideal), otherwise small apoptotic DNA bands will diffuse, resulting in poor resolution.
1. Collect cells, centrifuge 500 × g for 5 min at 4 °C. Wash cells twice with PBS. Collect 5-10 × 105 cells (ideally a control for non-apoptotic cells (control group) is included with the apoptotic cells (experiment group). Carefully pipette off the supernatant, remove as much hanging liquid drops on the tube wall as possible.
2. Flick to loosen the cell pellet at the bottom of the centrifuge tube, add 100 μl of Extraction Buffer. Vortex the mixture vigorously for 10 seconds. Centrifuge the tube(s) at 1,100-1,600 x g (3500-4,500 rpm) for 5 minutes.
3. Without disturbing pellet at the bottom of tube, transfer the supernatant to a new 1.5ml centrifuge tube.
4. For the pellet, repeat Step 2 once.
5. Combine the origina supernatant with the second supernatant, resulting in a total of approximately 200 μl crude extract (containing mostly apoptotic DNA fragments, as the non-apoptotic chromosomal DNA has been removed by precipitation).
6. Add 20μl of 10% SDS solution to the crude extract, add 20μl of Enzyme A, mix and incubate at 56 ℃ for 1 hour.
7. Add 20 μl of Enzyme B to the mixture, mix and incubate at 37 ° C for 1 hour, or until it becomes translucent (can leave the tube overnight at this temperature).
8. Add 130μl of Precipitant reagent (provided) to the mixture, invert and mix well, add 1 ml of ethanol, and mix well. Allow the mixture to stand for 1 hour or longer at 20 °C (to precipitate apoptotic DNA fragment).
9. Centrifuge tube(s) at 13,000rpm for 15min at 4ºC.Note the location on the tube where the pellet is expected. Tip: the pellet is expected at the distal part of the tube near the bottom. In order to avoid losing the pellet during this step, pour off the supernatant slowly.
Gently pour off the supernatant, rinse the pellet with 1ml of 70% ethanol, centrifuge again, slowly pour off ethanol, and pipette as much residual drops on tube wall as possible. Keep the lid open, air-dry precipitate in the tube at room temperature for 10 min.
10. Fully dissolve the pellet with 17 μl of double-distilled water or TE buffer by vortexing and a brief centrifugation. Mix the resuspended DNA with 3 μl of 6 × DNA gel loading buffer (not provided). Take the entire 20μl of the resulting sample to load a 1% agarose gels electrophoresis. A DNA marker should also be loaded along the sample DNA. Ethidium bromide or equivalent staining should be used to stain the DNA bands. UV or LED transilluminator or a gel documentation system can be used to observe and document the results. Tip: to compensate for low incidence of apoptosis, do not use volume of TE Buffer larger than recommended during elution; otherwise DNA concentration may be too low to clearly observe DNA ladders.