Kit Content
| Component | 100 Preps, PR242001 |
| GLB | 25 ml |
| TLB | 18 ml |
| BWB1 (concentrate) | 2 x 16 ml |
| BWB2 (concentrate) | 2 x 12 ml |
| ERR | 20 ml |
| RJ-Protease | 2 x 1.25ml |
| Carrier RNA | 620 μg |
| HiPure DR Column | 100 |
| Collection Tube | 200 |
Intended Use
DNall VirAll Kit provides the components and procedures necessary for purifying viral DNA/RNA from cell-free samples such as body fluid, serum, plasma and animal tissue. The assay is intended for in vitro diagnostic use. We recommend all users to study RNA experiments guideline, before starting their work.
Principle
DNall VirAll Kit is designed for isolating both viral DNA and RNA from body fluid, serum, plasma and genomic DNA from animal tissue. Lysis is achieved by incubation of the sample in GLB, for both DNA and RNA isolation; and in a RJ-Protease enzyme solution, just for viral DNA isolation. Appropriate conditions for DNA and RNA binding to the silica membrane is achieved by the addition of ethanol to the lysate. Then, DNA or RNA is selectively bound to the membrane. Contaminants are removed by two specific washing buffers. Pure viral DNA/RNA is finally eluted in rehydration buffer. Isolated DNA/RNA is ready to use in downstream applications. A symmetric peak at 260 nm by spectrophotometer, confirms high purity of isolated nucleic acid.
Sample Collection and Preparation
Sample Preparation
- If possible, use only fresh sample material. Do not freeze/thaw samples more than once.
- Plasma and serum samples can be stored at 2-8°C for up to 24 hours, or at −20°C or −70°C for long-term storage.
- Urine samples should be stored at 2-8°C for up to 12 hours (with 0.5 M EDTA added to 50 mM final concentration), or at −20°C or −70°C for long term storage (cells should be collected by centrifugation).
- Before use, equilibrate samples to room temperature (20±5°C). Remove precipitates from plasma/serum samples, if any, by centrifugation for 5min at 3,000 × g.
- Use EDTA or citrate treated plasma samples.
- Urogenital swabs can be stored at 2-8°C for up to 48 hours. For longer term storage cells should be collected by centrifugation and stored at −20°C or −70°C
- Nasal and buccal swabs can be stored at 2-8°C for up to 48 hours.
Washing Buffer Preparation
Before the first use, add appropriate amount of ethanol (96-100%) to each washing buffer tube, then mix thoroughly to prepare washing buffer, refer to the Table. Do not forget to tick the check box on the bottle label to indicate that ethanol has been added. Before each use mix reconstituted buffer by shaking. Storing at room temperature.
Table : Washing buffer preparation
| Buffer Name | Concentrated Volume | Amount of Ethanol | Final Volume |
| BWB1 | 16ml | 24ml | 40ml |
| BWB2 | 12ml | 28ml | 40ml |
Quality Control Procedure
DNall VirAll Kit is tested against predetermined experiments on a lot-to-lot basis according to ISO-certified quality management system, to ensure consistent product quality. For your information, the results of all experiments are accessible by addressing Cat and Lot number on web at https://amorph.tech.
Troubleshooting
Here we try to cover as many problems as you may see in using this product, however scientists in Carbon Technical Support Team are eager to answer all your questions. Do not hesitate to contact us for more information.
| Type | Symptoms | Problem | Suggestion |
| Viral DNA Isolation | Low DNA yield | Insufficient lysis | Please refer to Table 3 to apply best match for size of starting material and amount of lysis buffer. |
| Make sure to do pulse-vortexing after addition of lysis buffer and RJ-Protease. | |||
| Too few viruses in the sample | Do the test with new samples. | ||
| Incomplete lysing | Repeat the reaction once more and make sure to mix the sample and lysis buffer completely by pulse-vortexing. | ||
| Reagents not applied correctly | Prepare buffers according to the protocol. | ||
| Make sure ethanol is added to BWB1 and BWB2. | |||
| Repeat the procedure with a new sample. | |||
| DNA improperly eluted | The best buffer for DNA rehydration is prepared in the Kit Box. We insist to use the supplied rehydration buffer, however if you want to use water instead, make sure that the pH is at least 7.0, or use 10 mM Tris-HCl Ph≥ 7.0. | ||
| DNA does not perform well in downstream applications | DNA was not washed with the provided washing buffer | Ensure the column was washed once with prepared BWB1 and once more with prepared BWB2, respectively. | |
| Ethanol carryover | Ensure that the traces of ethanol before rehydration step is removed. | ||
| General Handling | Column clogging | Precipitates were not removed. | When using plasma samples, remove visible Cryoprecipitates by centrifugation for 5min at 3000 × g |
| Lysate not completely passed through the membrane | Centrifuge for 1min at full speed or until all the lysate has passed through the membrane. |
Specific Characteristics
| Features | Specifications |
| Elution volume | 20-200 µl |
| Technology | Silica technology |
| Main sample type | Body Fluid, serum, Plasma, Animal tissue |
| Processing | Manual |
| Sample amount | Plasma and serum: Up to 200 µlAll tissue kind expect spleen: up to 25 mg (Spleen up to 10mg) |
| Biomolecule isolation | DNA and RNA |
| Operation time per reaction | Less than 30 min (for viral DNA/RNA isolation)Less than 20 min (for whole blood, buffy coat, serum and plasma)Less than 2 h (for animal tissue and bacteria) |
| Typical yield | Varies |
| Carrier RNA | Provided in the kit |
| Enzyme | RJ-Protease |
Storage and Safety
Shipment condition is checked by Carbon Technologies. After arrival, all reagents should be kept dry, at room temperature. We suggest storing RJ-Protease at 2-8°C, and for routine use, it is recommended that you aliquot it to 100 µl volumes and storage at 2-8°C. Also, Carrier RNA is storable at room temperature before preparation. However, after adding ERR buffer, it is recommended that it must store at -20°C and be aliquoted to 100 µl volumes and avoid frequent freeze-thaw. When storage condition is as directed, all reagents are stable until expiration date, as indicated on the kit box.
