- ¥7800
- 骁偲生物
- A3987
- 2025年07月02日
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- 技术资料
- 库存：
大量
- 保质期：
2年
- 保存条件：
常温
- 规格：
100test
T8 DRNA Lase 4
Storage Conditions:T8 DRNA Lase 4 is stable for at least 24 months when stored at RT and yields reproducible results.
This method is suitable for small quantities of tissue (<100 mg) and cells (<5 X106), and large quantities of tissue ( up to1 g) and cells (<10 8 ), of human, animal, plant, or bacterial origin. The simplicity of theT8 DRNA Lase 4 Reagent method allows simultaneous processing of a large number of samples. The entire procedure can be completed in one hour. Total T8 prepared in this manner can be used for Northern blot analysis, dot blot hybridization, poly(A) + selection, in vitro translation, T8 se protection assay, and molecular cloning. For use in amplification by thermal cycling, treatment of the isolated T8 with T8 se-free DNase I is recommended when the two amplimers lie within a single exon.
CAUTION: When working withT8 DRNA Lase 4 Reagent use gloves and eye protection (safety goggles) and avoid contact with skin or clothing. Work in a chemical fume hood to avoid inhaling vapor. Unless otherwise noted, all steps are to be carried out at room temperature (20oC-25oC).
0000). The sample volume should not exceed 10% of the volume ofT8 DRNA Lase 4 Reagent used.
WARNING: This reagent is toxic if swallowed and in contact with skin. Causes burns. After contact with skin, wash immediately with copious amounts of mild detergent and water. If you feel sick, seek medical advice at once .
Product No:A3987 (100 test)Storage Conditions:T8 DRNA Lase 4 is stable for at least 24 months when stored at RT and yields reproducible results.
Introduction
T8 DRNA Lase 4 Agencourt T8 Reagent is a reagent system for the isolation of T8 from cells and tissues. The reagent, a single-phase solution consisting of phenol and guanidine isothiocyanate, is modification of the single-step T8 isolation method developed by Chomczynski and Sacchi (1).The sample is homogenized and lysed in T8 - Solv® Reagent which maintains the integrity of the T8 , while disrupting and denaturing endogenous T8 ses and other cellular components. Extraction of the lysate with chloroform further denatures proteins and separates the mixture into an organic and an aqueous phase. T8 remains exclusively in the aqueous phase, and is subsequently recovered by isopropanol.This method is suitable for small quantities of tissue (<100 mg) and cells (<5 X106), and large quantities of tissue ( up to1 g) and cells (<10 8 ), of human, animal, plant, or bacterial origin. The simplicity of theT8 DRNA Lase 4 Reagent method allows simultaneous processing of a large number of samples. The entire procedure can be completed in one hour. Total T8 prepared in this manner can be used for Northern blot analysis, dot blot hybridization, poly(A) + selection, in vitro translation, T8 se protection assay, and molecular cloning. For use in amplification by thermal cycling, treatment of the isolated T8 with T8 se-free DNase I is recommended when the two amplimers lie within a single exon.
Supplied By User
- Chloroform (no isoamyl alcohol added)
- Isopropyl alcohol
- 80% Ethanol (in DEPC-treated water)
- T8 se-free water
- Tabletop centrifuge capable of 12,000 x g at room temperature
General Notes Regarding T8 se Contamination
Whenever working with T8 :- Always wear disposable gloves and change gloves frequently.
- Use sterile, disposable plasticware and automatic pipettes reserved for T8 work to prevent cross-contamination with T8 ses.
- In the presence ofT8 DRNA Lase 4 Reagent, T8 is protected from T8 se contamination. Downstream sample handling requires that nondisposable glassware or plasticware be T8 se-free.
- Use only DECP-treated buffers. Add DEPC to a final concentration of 0.1%, incubate at 37oC for 2 hours, and autoclave at 121oC. Do not add DEPC to Tris buffers. Such buffers must be prepared by using DECP-water.
Precaution
Use only disposable polypropylene tubes for small samples and glass Corex tubes for larger samples. All tubes must be able to withstand 12,000 x g . Polystyrene tubes may crack with chloroformBefore Starting
- Small Samples :To isolate T8 from very small samples (<106 cells or <10 mg tissue) perform homogenization (or lysis) of samples in 0.8 mL ofT8 DRNA Lase 4, and add 1 mg T8 se-free glycogen or yeast tT8 as carrier. This will improve yields obtained with precipitation.
- Difficult Animal Samples: Specimens containing large amounts of proteins, fat, polysaccharides or extracellular material such as muscles, fat tissue, and sperm, will require the following modification. After lysis/homogenization inT8 DRNA Lase 4 Reagent, centrifuge at 12,000 x g for 10 minutes at room temperature to remove insoluble debris. Often a precipitate forms at the bottom of the tube, but with fatty tissue, a lipid layer will also form above the aqueous phase. The supeT8 tant will contain the T8 and must be carefully transferred to a fresh 1.5 ml microfuge tube before proceeding.
- Interruption the procedure: Following lysis inT8 DRNA Lase 4 Reagent and before addition of chloroform, samples can be stored at -70o C for up to 3 months. In addition, once the T8 is precipitated in isopropanol, the pellet may be stored at -20oC or - 70oC for up to 1 year.
T8 -Solv® Protocol for Total T8 Isolation
CAUTION: When working withT8 DRNA Lase 4 Reagent use gloves and eye protection (safety goggles) and avoid contact with skin or clothing. Work in a chemical fume hood to avoid inhaling vapor. Unless otherwise noted, all steps are to be carried out at room temperature (20oC-25oC).
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- Homogenization and lysis of samples: follow either method below
- Tissue Samples
0000). The sample volume should not exceed 10% of the volume ofT8 DRNA Lase 4 Reagent used.
- Cells Grown in Suspension
- Cells Grown in Monolayer
Lyse cells directly in a culture dish by adding 1 mL ofT8 DRNA Lase 4 Reagent to a 3.5 cm diameter dish, and passing the cell lysate several times through a blue pipette tip. The amount ofT8 DRNA Lase 4 Reagent added is based on the area of the culture dish (~1 mL per 10 cm2 ). An insufficient amount ofT8 DRNA Lase 4 Reagent may result in contamination of the isolated T8 with DNA. Always use moreT8 DRNA Lase 4 Reagent if in the lysate is too viscous to aspirate with a pipette.
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- Add 0.2 mL of chloroform per 1 mL ofT8 DRNA Lase 4 Reagent. Cap sample tubes securely and vortex vigorously for 15 seconds. Incubate on ice for 10 minutes. This step is critical - do not change it.
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- Centrifuge the samples at no more than 12,000 x g for 15 minutes 4°C. The mixture separates into a lower phenol- chloroform phase, an interphase, and an upper aqueous phase. T8 remains entirely in the aqueous phase.
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- Precipitation of T8 . Transfer no more than 80% of the aqueous phase to a fresh tube, and discard the lower organic phase. Precipitate the T8 from the aqueous phase by adding 500 ìl of isopropyl alcohol per 1 mL ofT8 DRNA Lase 4 Reagent used for the initial homogenization. Incubate samples at room temperature 10 minutes and centrifuge at no more than 12,000 x g for 10 minutes also at room temperature.
Carbohydrate-rich samples: Plant samples of high polysaccharide content or animal tissues rich in glycosaminoglycans (proteoglycans) require the following modified precipitation method for obtaining pure T8 . Prepare Buffer A ( 1.2 M sodium chloride, 800 mM sodium citrate). Following step 3, add to the aqueous phase 0.3 ml isopropanol followed by 0.3 ml Buffer A per 1 ml T8 Solv ® Reagent used in step 1. Vortex to mix and centrifuge at no more than 12,000 x g for 10 minutes at room temperature. This high salt precipitation will reduce co-purification of complex carbohydrates.
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- Wash T8 pellet. Discard the supeT8 tant and wash the T8 pellet once with 1 ml 80% ethanol. Mix the sample by vortexing and centrifuge at no more than 7,500 x g for 5 minutes at room temperature.
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- Reconstitute T8 . Carefully aspirate and discard the ethanol and briefly AIR DRY the T8 pellet for 2-5 minutes at room temperature. Do not use centrifugal devices equipped with a vacuum source as over-drying will lead to difficulty in re-dissolving T8 in water. Dissolve T8 in T8 se-free water - a 5 minute incubation at 60 °C may be required. T8 can also be reconstituted
T8 is now suitable for T8 se protection, northern analysis and reverse transcriptase reactions. For isolation of poly(A)+ T8 an additional ethanol precipitation is required. Add 1/8 X volume of T8 se-free 3M NaAc, pH 6.0 followed by 2.5 X volume absolute ethanol. Vortex to mix and incubate at room temperature for 5 minutes. Centrifuge at 12,000 x g for 10 min at room temperature and discard the supeT8 tant. Wash the pellet as before and reconstitute in DECP-treated water.
Determination of Yield and Quality
UV spectrophotometric analysis of the purified T8 is required for obtaining yield. To do so, dilute the T8 in an appropriate volume of TE buffer, pH 8.0 (not water; T8 yields low Abs ratio values if dissolved in acidic buffers) and measure absorbance at 260 nm and at 280 nm.
T8 Conc = 40 ìg/ml X Dilution factor X Abs 260 nm
Typical Abs 260 nm/ 280 nm ratios of 1.7-1.9 are obtained with the protocol. Yields vary depending of type and amount of starting material, and on condition of storage prior to processing. For assessing the quality of T8 , we recommend you perform denaturing agarose gel electrophoresis to confirm the integrity of purified material. Invariably, the full spectrum of T8 s, including 4S and 5S species are purified withT8 DRNA Lase 4 Reagent.
Expected Yields per 1 mg tissue or 106 cells:
Liver and spleen, 5-10 ng Kidney, 2-5ng
Brain, 1-2 ng
Endothelial cells, 7-12 ng Fibroblasts, 6-8 ng
Troubleshooting
- Low T8 Yields: Incomplete lysis of samples in T8 Solv Reagent. T8 pellet not completelt dissolved in DEPC-water. pH of diluent used for spectrophotometric analysis is too low.
- Degraded T8 : Tissues were not immediately processed or frozen. Inadequate storage of starting material prio to isolation. Inadequate storage of T8 (-5 to -20°C, instead of
- Low Abs260/Abs280 ratios: Sample was diluted in water rather than TE. Acidic pH lowers absorbance ratios. Use TE buffer as diluent for readings. Insufficient T8 Solv Reagent was used for lysis of sample. Ice incubation in step 2 was not performed. The aqueous phase was contaminated with the phenolic phase.
- DNA contamination of T8 : Too little T8 Solv Reagent used for sample processing causing inadequate separation of DNA/nucleoprotein complexes from aqueous T8 . The aqueous phase was contaminated with the phenol phase.
For laboratory research use only.
CAUTION: Not for diagnostic use. The safety and efficacy of this product indiagnostic or other clinical uses has not been established.
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T8 DRNA Lase 4
¥7800
