Navigation Links
Total RNA Isolation: 100 l Viral Sample From Cultured-Cell Supernatant Containing Virus


AquaPure RNA Isolation Kit
Catalog #732-6370
Yield: Dependent on sample and RNA expression levels


Method
Sample Collection and Handling
Samples may be fresh or frozen. Collect fresh virus and isolate RNA as quickly as possible; store at 18C for not more than 24 hr. For long-term storage, store at -70 to -80C.

Cell Lysis
1. Add 500 l cell lysis solution to a 1.5 ml microfuge tube that contains 100 l virus from cultured-cell supernatant.

2. Cap the tube and vortex for 5 sec to mix thoroughly.

3. Heat sample to 65C for 5 min to complete lysis. Cool sample to room temperature. (This step is optional when testing for certain viruses such as hepatitis C.)

Protein-DNA Precipitation
1. Add 200 l protein-DNA precipitation solution to the viral cell lysate.

2. Invert capped tube gently 10 times and place tube into an ice bath for 5 min.

3. Centrifuge at 13,00016,000 x g for 3 min. The precipitated proteins and DNA will form a tight pellet.

RNA Precipitation
1. Leaving behind the precipitated protein-DNA pellet, pour the supernatant containing the RNA into a clean 1.5 ml microfuge tube containing 600 l 100% isopropanol (2-propanol). If RNA yield is expected to be low (<1 mg), add glycogen as a carrier to the isopropanol. We recommend adding 1 l of glycogen solution (20 mg/ml) per 600 l isopropanol.

2. Cap the tube and mix the sample by inverting gently 50 times.

3. Centrifuge at 13,00016,000 x g for 3 min; depending on yield, the RNA may or may not be visible as a small, translucent pellet.

4. Pour off supernatant and drai n tube on clean absorbent paper. Add 600 l 70% ethanol and invert the capped tube several times to wash the RNA pellet.

5. Centrifuge at 13,00016,000 x g for 1 min. Carefully pour off the ethanol.

6. Invert and drain the tube on clean absorbent paper and allow to air-dry 1015 min.

RNA Hydration
1. Add 20 l RNA hydration solution (20 l will give a concentration of 50 g/ml if the total yield is 1 g RNA).

2. Allow RNA to rehydrate on ice for at least 30 min. Store purified RNA sample at 70 to 80C until use.

3. Before use, vortex sample vigorously for 5 sec and pulsespin. Pipet sample up and down several times to ensure adequate mixing.



back to top
'"/>

Source:


Page: All 1 2

Related biology technology :

1. New Kit Generates Exceptionally Pure Total RNA for RT-PCR
2. High-Throughput Isolation of Total RNA
3. Isolate and Analyze Total RNA from Cells Harvested by Laser Capture Microdissection
4. Fast Isolation of Total RNA from Small Numbers of Cells
5. Total RNA Isolation Protocol 1,2,3
6. Isolate It All: siRNA miRNA Total RNA Native Protein
7. Isolate Total RNA and Protein From the Same Sample
8. Isolate High Quality Total RNA from LCM Samples Suitable for Microarray and qRT-PCR Analysis
9. Recover High Yields of Total Nucleic Acid from Formalin-fixed, Paraffin-embedded (FFPE) Tissue
10. Total RNA from Whole Blood for Expression Profiling
11. RNAPrep - Total RNA Isolation Purification
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... 27, 2016  Liquid Biotech USA ... of a Sponsored Research Agreement with The University ... (CTCs) from cancer patients.  The funding will be ... correlate with clinical outcomes in cancer patients undergoing ... then be employed to support the design of ...
(Date:6/24/2016)... , ... June 24, 2016 , ... While the majority ... as the Cary 5000 and the 6000i models are higher end machines that use ... height of the spectrophotometer’s light beam from the bottom of the cuvette holder. ...
(Date:6/23/2016)... Mass. , June 23, 2016   ... development of novel compounds designed to target cancer ... napabucasin, has been granted Orphan Drug Designation from ... the treatment of gastric cancer, including gastroesophageal junction ... stemness inhibitor designed to inhibit cancer stemness pathways ...
(Date:6/23/2016)... ... June 23, 2016 , ... Charm Sciences, Inc. ... test has received AOAC Research Institute approval 061601. , “This is another AOAC-RI ... stated Bob Salter, Vice President of Regulatory and Industrial Affairs. “The Peel Plate ...
Breaking Biology Technology:
(Date:6/20/2016)... -- Securus Technologies, a leading provider of civil ... investigation, corrections and monitoring announced that after exhaustive ... the final acceptance by all three (3) Department ... (MAS) installed. Furthermore, Securus will have contracts for ... October, 2016. MAS distinguishes between legitimate wireless device ...
(Date:6/9/2016)...  Perkotek an innovation leader in attendance control systems is proud to announce the ... employers to make sure the right employees are actually signing in, and to even ... ... ... ...
(Date:6/3/2016)... 3, 2016 Das ... Nepal hat ein 44 ... geprägter Kennzeichen, einschließlich Personalisierung, Registrierung und IT-Infrastruktur, ... Produktion und Implementierung von Identitätsmanagementlösungen. Zahlreiche renommierte ... Januar teilgenommen, aber Decatur wurde als konformste ...
Breaking Biology News(10 mins):