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FAQ Apoptosis Ladder Detection Kit Wako
(Wako Catalog No.297-71401)

Characteristics of the kit

Q: How much is the yield of DNA?
A: DNA can be extracted, in a yield more than 90 %.
Q: What is the purity of DNA obtained?
A: A purity of DNA as high as OD260/280 ≒1.9 can be obtained.
Q: What is the range of number of cells which can be analyzed?
A: It is 103 ~ 106. Less than 103 is below the limit of detection, while more than 107 will cause the viscosity of recovered DNA too high to be applied to Agarose Gel.
Q: How is the apoptosis among tissue cells detected?
A: Detection may be feasible from frozen slices samples from unfixed fresh tissue by applying preliminary treatment with methods suitable for individual tissues, such as homogenization.
Q: What other materials must be prepared in addition to the kit?
A: Reagents needed are Isopropanol, 70% Ethanol, TAE Buffer, Agarose S, and sterile distilled water. Also needed will be 1.5 mL microtube, centrifuge, casting stand, gel tray, comb, submarine-type electrophoretic device, plastic vessel, shaker, UV transilluminator, camera, and Yellow Gelatin Filter (SYBER® Green/Gold Gel Photographic Filter)

Extraction of DNA

Q: When should the reagents stored at -20℃ be thawed, such as RNase, Enzyme Activator, Protein Digestion Enzyme and SYBR® Green I?
A: These shall be thawed at room temperature immediately before their use.
Q: What is the range of number of specimen cells?
A: The range is 103 ~ 106, but the most clearly visible images of electrophoresis of DNA ladder can be obtained from 104 cells.
Q: Is 30 minutes enough for proteolysis (protein decomposition)?
A: Normally, 50℃ for 30 minutes will be sufficient. If any traces of pellets are left yet to be decomposed, the proteolytic reaction should be continued until they are completely dissolved. In the case of cells that are difficult to dissolve, temperature should be elevated to approx. 56℃, with frequent tapping to facilitate reaction.
Q: What if a large amount of RNA is mingled in the specimen?
A: For such cases, Rnase reaction should be continued for a longer period of time, or otherwise, the extracted DNA should be dissolved into TE Buffer and subjected to RNase treatment (with final concentration of 10 μg/mL for 1 hour at 37℃ or at 4℃ overnight).

Electrophoresis

Q: What kind of electrophoretic devices can be used?
A: The submarine-type electrophoretic device, such as Mupid can be used.
Q: How much amount of DNA sample should be applied to the Agarose Gel?
A: When the DNA sample is in an amount of 103 ~ 104 cells, the whole amount of 10 μL of TE Buffer in which the DNA is dissolved should be applied. For other cases, the amount of DNA should be measured and its amount or sample should be adjusted into 10 ~ 100 ng/well and then applied. DNA which can be obtained from cells with low apoptosis induction rate has such a high viscosity that it can hardly be applied to a well.
Therefore, it should be diluted into more TE Buffer into the applicable concentration.
Q: What is the time needed for electrophoresis?
A: It is 30 - 40 minutes. The standard for it will be the time BPB, which shows the higher speed for electrophoresis of all the pigments in the Loading Buffer, reaches approx. 1cm before the end of anode of the gel.
Q: How is the TAE Buffer to be prepared?
A: Either 50 x stock should be prepared according to the following composition, or 50 x TAE (Wako Cat. No.313-90035; 500 mL) should firstly be prepared, which should be diluted freshly into 50 time volume before use. The 50 x stock solution can be prepared with Tris Base 242 g, Acetic Acid 57.1 mL and 0.5 M EDTA 100 mL (pH 8.0)
Q: Can TBE be used for the Electrophoresis Buffer?
A: When the agarose gel is made with TAE Buffer, TBE Buffer cannot be used. If TBE Buffer will be used, prepare the agarose gel with TBE Buffer.

Staining with SYBER® Green

Q: Is the attached SYBR® Green I identical with that of Molecular Probes, Inc.?
A: It is a product of Molecular Probes, Inc. It is licensed by this company as a component for the kit.
Q: What is the difference between the SYBR® Green I and EtBr?
A: As is the case of EtBr, SYBR® Green I is an intercalater of nucleic acids. It is characterized by 25 times higher detection sensitivity and lower mutagenicity than those of EtBr.
Q: What are the precautions for handling SYBR® Green I?
A: As it is a solution in DMSO, wearing two pairs of protective glove is necessary for its handling.
Q: How should SYBR® Green I be stored?
A: After opening the kit, SYBR® Green I should be fully stirred with a Vortex mixer and approx. 12 μL aliquots should be distributed into polypropylene tubes. These should be stored at -20℃ with protection from light and should be quickly thawed into room temperature just before use. Glass containers or other types of containers can cause stability loss during storage because these will absorb the pigments.
Q: What measured should be taken if any precipitates are formed in the SYBR® Green I?
A: It should be sufficiently stirred with a Vortex mixer until the precipitates are dissolved and then it should be used.
Q: How should the SYBR® Green I be diluted?
A: It should be diluted with TAE Buffer into a concentration of 1:10,000. If it is overly diluted, its detection sensitivity will be deteriorated. Its sensitivity will be also reduced, if it is diluted with distilled water.
Q: What is the stability of the diluted SYBR® Green I?
A: It is 24 hours when stored with protection from light at room temperature or at 4℃.
Q: What types of containers can be used for staining with SYBR® Green I?
A: Staining trays for which are made of polypropylene should be used.
Q: How long is needed for staining with SYBR® Green I?
A: It should be a minimum of 30 minutes. If stained for less than 30 minutes, its detection sensitivity will be reduced. Its sensitivity will remain unchanged while time of staining is prolonged, up to 120 minutes.
Q: What is the disposal procedure for SYBR® Green I?
A: As is the case of EtBr, it should be filtered through activated carbon for absorption of SYBR® Green I, and then the activated carbon after its absorption should be disposed of as hazardous waste sealed in a plastic bag (Ref.: Molecular Cloning, A Laboratory Manual, 2nd ed., E9), EXTRACTOR can be used as the filter for activated carbon.
Q: What is the wave length of light used to visualize DNA stained with SYBR® Green I?
A: Shorter wave lengths are more effective for this purpose, with 254 nm yielding the most sensitive results. Also, 300 nm will give almost equal sensitivity.
Q: How long can the Agarose Gel stained with SYBR® Green I be illuminated?
A: The time for excitation by UV irradiation must be less than 1 minute. If it exceeds 1.5 minutes, its detection sensitivity will be drastically reduced. The degree of such reduction depends on the time of irradiation. Once the intensity of fluorescence is reduced (i.e. the detection sensitivity is reduced), the intensity of fluorescence can not be restored even with UV irradiation after re-staining with SYBR® Green I or even with re-irradiation by UV.
Q: Can the background for UV detection of Agarose Gel stained with SYBR® Green I be lowered by washing with the Buffer or distilled water?
A: The background will not be altered even with the washing. For lowering the background, Yellow Gelatin Filter exclusive for such use should be used.
Q: What are the conditions for photographing the gel after staining with SYBR® Green I?
A: In the case of Polaroid camera, it is recommended to use a shutter speed of 1/2s, an iris of 4,5, and a film sensitivity of ASA 3200. In the case of single lens reflex, it should be with a shutter speed of 1/4s, an iris of 2.8 and a film sensitivity of ASA 1600.
Q: How should the Yellow Gelatin Filter (SYBR® Green/Gold Gel Photographic Filter) be used?
A: It should be held in front of the camera lens in order to lower the background in taking an electrophoretic photograph. If a holder exclusive for its use is available, it should be used. Also, Red Gelatin Filter (for EtBr) can be used, but, in order to obtain an even better sensitivity, the Yellow w Gelatin Filter should be used.

Trouble shooting

Q: What measured should be taken if crystals are formed in the Enzyme Reaction Solution?
A: Crystals may form at a lower temperature. For such cases, the tube should be placed in water heated at 50℃ until crystals dissolve, and then cooled down to room temperature before it use.
Q: What measured should be taken if the extracted DNA can be dissolved into TE Buffer?
A: "If the pellet DNA is made too dry, it will be hard to dissolve. For such cases it should be dissolved for a long period of time (such as overnight). The dissolution can be also facilitated by further addition of TE Buffer. In such case, mixing the Buffer with a pipette should be avoided that may damage DNA."
Q: What measures should be taken if sufficient detection sensitivity is not obtained?
A: The detection limit of this kit is 103 apoptosis cells. Therefore, with specimens which have less than 100% apoptosis induction, the apoptosis cells can not be detected with initial amount of 103 cells, which should be therefore increased up to 104 - 106 cells.
Q: What measures should be taken if DNA ladder is detected with negative control?
A: It may be attributed to the overly accelerated conditions for DNA extraction. For dissolving DNA, stirring with Vortex mixer or rigid pipetting should be avoided, and tapping with fingertips should be adopted for gentle dissolution. As the negative control is not included in the kit, it should be made by each user.
Q: What measures should be taken if the band of the Ladder Marker is too vague or hardly visible on the electrophoretic photograph?
A: If the exposure of the camera is insufficient, it may cause dimness of the bands of Ladder Marker or DNA samples.
For such cases, the shutter speed and iris of the camera should be properly adjusted to obtain enough exposure for photographing.
Q: What measures should be taken if the pellets are hard to peel off the inner wall of the tube during wash with ethanol?
A: The tubes should be tapped rather strongly with fingertips. The mixing with Vortex mixer or rigid pipetting should be avoided to prevent damage of DNA.

Other

Q: What are the precautions for interrupting the operation for some reasons?
A: The interruption is acceptable when the extracted DNA is dissolved into TE Buffer and placed into cold storage.
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