Download RnaUs Total Viral RNA/DNA Prep

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RnaUs Total Viral RNA/DNA Prep
Catalog No. 7322-05: RnaUs Total Viral RNA/DNA Prep, Size: 50 Reactions
Catalog No. 7322-25: RnaUs Total Viral RNA/DNA Prep, Size: 250 Reactions
Description
LeGene RnaUs Total Viral RN A P r e p provides an easy and reliable method for isolating total viral
RNA from plasma or serum while enzyme inhibitors and other contaminates completely removed. This
procedure has been validated for isolating nucleic acids from Hepatitis A, Hepatitis B, Hepatitis C and
HIV. This kit can also be used to isolate viral DNA and viral RNA from urine and cell culture supernatant.
The purified RNA/DNA is of the highest integrity, and can be used in a number of downstream
applications including real time RT-PCR, cDNA synthesis, Northern blotting, RNase protection and
primer extension, miRNA cloning and amplification, and next generation RNA sequencing.
The binding capacity of each ezCapture RNA Micro Column is approximately 10 g RNA/DNA and
recommend to use up to 300 l plasma or serum.
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Storage and Stability
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Store RNA carrier at 4 C. All other components can be stored at room temperature. Kit components are
guaranteed for 12 months from the date of purchasing.
Kit Contents
Catalog#
7322-05
# of Preps
50
7322-25
250
Buffer P
28 ml
130 ml
Buffer G
28 ml
130 ml
RNA Wash Buffer
12 ml
50 ml
RNA Carrier
280 l
10 ml
1000 l
30 ml
RNA Elution Buffer
ezCapture RNA Micro Columns
50
250
Collection Tubes
100
500
User Instruction
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Safety Information
Buffer P contains chaotropic salts, which may form reactive compounds when combines with bleach.
Do not add bleach or acidic solutions directly to the preparation waste, wear gloves and protective
eyewear when handling.
LeGene Biosciences Inc. Web:www.legenebiosciences.com
E-mail:[email protected], Tel: 1-888-420-2220
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Check List Before Start
Prepare all components and get ready the necessary materials by reviewing the provided
instruction guidelines.
Important
Add 48 m (7322-05) or 200 ml (7322-25) 100% ethanol to RNA Wash Buffer before use. The final
ethanol is 80% (v/v).
Materials supplied by users
Tabletop microcentrifuge
1.5 ml sterile tubes
100% ethanol.
Perform all steps including centrifugation at room temperature.
LeGene Biosciences Inc. Web:www.legenebiosciences.com
E-mail:[email protected], Tel: 1-888-420-2220
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Protocol For Total Viral RNA Extraction From Plasma or Serum
1. Pipet 100-300 l plasma or serum into a 1.5 ml tube and add 2 volumes of Buffer P into the
plasma or serum.
2. Mix it thoroughly by vortexing for 2 seconds.
3. Add 0.5 volume 100% ethanol into the lysate (for example: 250 l o f 100% ethanol for 500 l
lysate) and mix the solution by pipetting (5-6 times). Add 5 ul of RNA Carrier, mix well by vortexing for
2 seconds.
4. Transfer the solution into an e zC a p t u r e RNA Mic r o Column and centrifuge at 12,000 rpm for 1
min. Discard the collection tube with the flow-through and put the column back to a new
collection tube.
5. Add 500 l Buffer G to the column and centrifuge at 12,000 rpm for 30 s. Discard the flow-through.
6. Add 500 l RNA Wash Buffer to the column and centrifuge at 12,000 rpm for 1 min. Discard the
flow-through.
Note: Ensure that ethanol has been added to RNA Wash Buffer before use.
7. Add another 500 l RNA Wash Buffer to the column and centrifuge at 12,000 rpm for 30 s.
Discard the flow-through and put the column, with the lid open, back to the collection tube.
8. Centrifuge at 14,000 rpm for 1 min. Discard the flow-through.
9. Centrifuge the empty column, with lid open, at 14,000 rpm for 1 min.
Note: It is critical to remove residual ethanol for optimal elution.
9. Place the column to a RNase-free 1.5 ml tube, add 15-30 l RNA Elution Buffer to the column
and centrifuge at 12,000 rpm for 2 min. The RNA is in the flow-through liquid. Store the RNA solution
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at -20 C.
Note: It is highly recommended that RNA quality be determined before downstream applications.
The quality of RNA can be assessed by denatured agarose gel electrophoresis with the ethidium
bromide staining. Several sharp bands should appear on the gel including 28S and 18S
ribosomal RNA bands as well as certain populations of mRNA and bands. If these bands
smear towards lower molecular weight RNAs, then the RNA has undergone major degradation
during preparation, handling or storage. An A260/A280 ratio of 1.8-2.0 corresponds to 90-100%
pure nucleic acid.
Trouble Shooting Guide
Problems
Possible reasons
Protein contamination
Suggested Improvements
Perform Phenol:Chloroform extraction. Loss of
total RNA i s expected (up to 40%).
LeGene Biosciences Inc. Web:www.legenebiosciences.com
E-mail:[email protected], Tel: 1-888-420-2220
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Low A260/A280 Guanidine Thiocyanate
contamination
ratios
Low Yield
Add 2.5 volumes of ethanol and 0.1M NaCl (final
concentration) to precipitate RNA. Incubate for 30
min at –20°C. Centrifuge at 13,000 rpm for 15 min
at 4°C.Resuspend the RNA pellet in DEPC-treated
water.
RNA in sample degraded
Freeze samples immediately in liquid nitrogen and
store at -80°Cafter collect it.
The binding capacity of the
membrane in the spin
column was exceeded
Use of too much sample exceeding the binding
capacity of spin column will cause the decreasing
of total RNA yield.
Ethanol not added to buffer
Add ethanol to the RNA Wash Buffer before
purification.
Limitations of Use
For research use only. Not for use in diagnostic procedures.
LeGene Biosciences Inc. Web:www.legenebiosciences.com
E-mail:[email protected], Tel: 1-888-420-2220
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