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Product Information
| Product Name | Cat.No. | Spec. |
| Animal Tissue Direct PCR Kit | G3310-100T | 100 T |
| G3310-500T | 500 T |
Description
This product is a kit specially designed for direct PCR amplification of animal tissues. Applicable samples include fresh or frozen mammalian viscera, nails, hair, zebrafish, drosophila, cultured cells, etc. The unique Animal Tissue Lysis Buffer A in the kit can be combined with Animal Tissue Lysis Buffer B to quickly release genomic DNA in micro tissues, without genomic extraction and purification, and can be directly used as a template for PCR amplification. It is simple to operate, reduces sample loss, and greatly shortens the experimental time. Pyrolysis products can be stored at – 20℃ for a long time.
2×Animal Tissue Direct PCR Mix (+Dye) in this kit contains all components except templates and primers. DNA Polymerase has the ability to rapidly amplify and resist inhibitors after modification. PCR Mix also contains a variety of reaction enhancers, which can further improve the amplification efficiency and specificity of DNA Polymerase, and can effectively amplify target genes within 3 kb.
Storage and Handling Conditions
Ice bag transportation; Animal Tissue Lysis Buffer A, stored at 4℃; Other components shall be stored at -20℃; Valid for 12 months.
Component
| Component Number | Component | G3310-100 | G3310-500 |
| G3310-1 | Animal Tissue Lysis Buffer A | 5 mL | 25 mL |
| G3310-2 | Animal Tissue Lysis Buffer B | 200 µL | 1 mL |
| G3310-3 | 2×Animal Tissue Direct PCR Mix (+Dye) | 1 mL | 5×1 mL |
| Product Manual | 1 | ||
Assay Protocol
1. Splitting the sample to release genomic DNA
a) Recommended sample dosage
| Class | Volume |
| Animal visceral tissue | ≤10 mg |
| hair | ≤10 mg |
| finger nail | ≤10 mg |
| Animal cells | ≤103cells |
b) Preparation of cracking liquid：
| Component | Volume (Single Sample) |
| Animal Tissue Lysis Buffer Aa | 50 µL |
| Animal Tissue Lysis Buffer B | 2 µL |
a. Animal Tissue Lysis Buffer A has white precipitates under low temperature. Incubate at 37℃ for 2-3 min before use until the white precipitates disappear.
c) Mix gently and centrifuge instantaneously；
d) Weigh the sample according to the recommended sample dosage and place it in a 1.5mL EP tube. Add the lysate required for a single sample into the tube to ensure that the tissue is immersed in the lysate. Incubate the centrifuge tube at 50℃ for 10min, and then at 95℃ for 3min；
e) Centrifuge at 12000 rpm for 1 min, centrifuge the cracked residue to the tube bottom, and the supernatant can be directly used as the PCR reaction template. The supernatant can be transferred to another sterile EP tube and stored at – 20℃ for a long time.
2. Recommended PCR reaction system (20 μL)：
| Component | 20 μL rxn | Final Concentration |
| Templatea | 1-2 μL | as required |
| Forward Primer (10 μM)b | 1 μL | 0.5 μM |
| Reverse Primer (10 μM)b | 1 μL | 0.5 μM |
| 2×Animal Tissue Direct PCR Mix (+Dye) | 10 μL | 1× |
| ddH2O | Add to 20 μL |
a The dosage of formwork shall not exceed 1/10 of the reaction system.
b：Use range of primer final concentration 0.5-1.0 μM. The recommended primer concentration is 0.5 μM. Too few primers may lead to low amplification yield or no amplification, and too many primers may lead to non-specific amplification.
3. Recommended PCR amplification conditions：
| Step | Temperature | Time | Cycles |
| Initial Denaturation | 98℃ | 2 min | 1 |
| Denaturation | 98℃ | 15 s | 35 |
| Annealing | 50-65℃ | 20 s | |
| Extension | 72℃ | 10 s/ kb | |
| Final extension | 72℃ | 5-10 min | 1 |
| Hold | 4-16℃ | Forever |
Note:
1. Before using the reagent, please gently mix it upside down. Do not swirl and oscillate to avoid bubbles.
2. Repeated freezing and thawing of frozen tissue should be avoided, otherwise the genome will be degraded and the PCR amplification effect will be affected.
3. Animal Tissue Lysis Buffer A and Animal Tissue Lysis Buffer B should be used in combination, as too long will affect the Lysis effect.
4. For samples that are difficult to crack, such as nails, hair, shell, etc., the cracking time can be extended to 20 min at 50℃.
5. Before lysis of intestinal tissue, use ddH2O to clean the surface impurities, and the amplification effect is better.
6. After lysis, incomplete lysis tissue remains in EP tube, which is a normal phenomenon and does not affect the use.
For Research Use Only!
Ver. No.: V1.0-202111
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