- Componenti del kit di reagenti
| Specifiche | 50T | 100T |
| Gatto. NO. | SN0205 | SN0206 |
| Colonne di estrazione del DNA (impostato) | 50 (impostato) | 100 (impostato) |
| Reagent Buffer I | 20 ml | 2×20 ml |
| Reagent Buffer II | 15 ml | 15 ml |
| Tampone reagente C | 30 ml | 2 × 30 ml |
| Tampone di eluizione 1 | 15 ml | 2 × 15 ml |
| Tampone di lavaggio | 20 ml | 20 ml |
| RNasi A | 1ml | 1ml |
| Manuale di istruzioni | 1 | 1 |
- Magazzinaggio
This kit should be stored at room temperature (15-25℃) in dry conditions and can be stored for 12 mesi. DNA extraction purification columns can be stored in a cool and dry environment for up to 1 year. RNase A contains a preservative and can be transported at room temperature, ma per la conservazione a lungo termine, it should be kept at -20℃.
- Istruzioni per l'uso del kit di reagenti
3.1 This kit is intended for molecular biology research purposes and should not be used for disease diagnosis or treatment.
3.2 Some components in the kit contain irritants; it is advisable to take necessary precautions (such as wearing protective clothing and goggles).
3.3 The use of this kit requires additional equipment such as a high-speed centrifuge, bagnomaria (bagno di metallo), miscelatore a vortice, etanolo anidro, liquid nitrogen, chloroform, acqua deionizzata sterile, and EP tubes.
- Introduzione al kit di reagenti
The new plant genome DNA purification kit provides a rapid method for DNA purification. It effectively precipitates DNA with specific reagent buffers II and C, collecting high-purity DNA through adsorption columns.
This kit is widely applicable for isolating samples from plant tissues and fungi, enabling the extraction of total DNA from plants and fungi within 30 minuti. The extraction process doesn’t involve toxic reagents like phenol-chloroform. The extracted DNA can be directly used for downstream experiments such as PCR and Southern blotting.
- Principi e procedure sperimentali：
- Processo di estrazione
Precautions before starting the experiment:
UN. Reagent Buffer I and Reagent Buffer C tend to precipitate at low temperatures. It is recommended to heat them at 65°C for 5 minutes until the precipitates dissolve before normal usage.
B. Before using Tampone di lavaggio 1, add the specified amount of absolute ethanol as indicated on the reagent bottle label and mark a check (√) on the label to indicate the addition of absolute ethanol.
C. The Elution Buffer is a 0.1x soluzione TE contenente una quantità minima di EDTA. If EDTA affects subsequent experiments, it is recommended to substitute the Elution Buffer with sterile deionized water.
1. Elaborazione del campione:
UN. Material Collection and Storage
If freshly collected materials cannot be immediately used, place them in liquid nitrogen for cooling and finally store at -80°C. Dried materials can be stored at room temperature.
B. If conditions permit, collect fresh materials whenever possible, as fresh materials contain fewer polysaccharides and polyphenols.
C. When collecting fungi from liquid culture, separate the liquid and collect the fungal cells by centrifugation.
2. Weigh around 100 mg of fresh samples or not exceeding 20 mg of dry material and grind it with liquid nitrogen.
(Nota: Different sample amounts may vary; it is advisable to optimize the sample amount through pre-experimental trials.)
3. Aggiungere 400μl Reagent Buffer I and 10μl RNaseA (10 mg/ml), ensuring there are no clumps in the ground sample. Clumps are difficult to lyse, reducing the DNA yield. Anche, do not mix Reagent Buffer I and RNaseA before usage.
4. Incubate at 65°C for 5 minuti, gently invert the mixture 2-3 volte. This step is used for cell lysis. If samples are difficult to lyse, extend the incubation time, but not beyond 30 minuti.
5. Aggiungere 130μl Reagent Buffer II and mix well, then ice-bathe for 5 minuti (this step is used for precipitating polysaccharides and proteins).
6. Centrifuge the lysate for 5 minutes at 14,000 giri/min (20,000× g).
(Nota: Some plant materials may contain a lot of viscous substances at this step, which can shear DNA in the subsequent steps. Perciò, the ideal state is to remove these substances during this step. Dopo la centrifugazione, transfer the supernatant to a new centrifuge tube. If there is a significant amount of flocculent material in the supernatant after centrifugation, it indicates that the initial sample quantity was too large. Consider reducing the initial sample amount.)
7. Carefully transfer the obtained liquid to a new centrifuge tube.
(Nota: Approximately 450μl of liquid can be transferred; for some species, it may be less than 450μl.)
8. Aggiungere un volume uguale di Tampone reagente C and an equal volume of absolute ethanol to the lysate and mix well.
(Per esempio: Add 450μl of Reagent Buffer C, then add 450μl of absolute ethanol. If the volume of the lysate is less than 450μl, reduce the amount of Reagent Buffer C proportionally. Adding Reagent Buffer C will cause slight precipitation, but it won’t affect subsequent experiments.)
9. Add the obtained liquid to the DNA extraction purification column (kit) (circa 650-700μl ogni volta), centrifuge at greater than 8,000 giri al minuto per 1 minuto, scartare i rifiuti raccolti, e reinserire il tubo di raccolta nella colonna di purificazione per il passaggio successivo.
10. Ripeti il passaggio 9, add the remaining liquid to the DNA extraction purification column (kit), centrifuge at greater than 8,000 giri al minuto per 1 minuto, discard waste and the collection tube.
11. Posizionare la colonna di purificazione per l'estrazione del DNA (kit) nel tubo di raccolta, aggiungere 300ml di tampone di lavaggio 1,centrifuge at greater than 8,000 giri al minuto per 1 minuto, discard waste, e posizionare la colonna di purificazione per l'estrazione del DNA (kit) nuovamente nel tubo per il passaggio successivo.
(Nota: Confirm the addition of absolute ethanol in Wash Buffer 1.)
12. Aggiungere 500ml di tampone di lavaggio 1 alla colonna di purificazione per l'estrazione del DNA (kit), centrifugare a 14,000 giri/min (20,000× g) per 2 minuti, extend centrifugation time appropriately to ensure the membrane is adequately dry.
13. Posizionare la colonna di purificazione per l'estrazione del DNA (kit) in una nuova provetta da centrifuga, leave it uncovered, and incubate at 65°C for 2 minuti. Extend this step as needed to evaporate ethanol to prevent ethanol residue from affecting downstream experiments.
14. Pipetta 100μl di tampone di eluizione onto the column membrane, centrifugare a 12,000 giri al minuto per 2 minuti.
(Nota: 1. Using 50μl of Elution Buffer to elute DNA can increase DNA concentration but reduce the overall DNA yield; 2. The eluate containing DNA can be reapplied to the DNA extraction purification column and centrifuged at 12,000 giri al minuto per 2 minutes again to increase DNA yield.)
Recensioni
Non ci sono ancora recensioni.