DIAKEY® REALcheck Viral DNA/RNA Prep Kit

[For Column Type]

General Description
DIAKEY REALcheck Viral DNA/RNA Prep Kit is a safe kit to quickly extract viral RNA / DNA from various samples such as serum, plasma, cell-free body fluids, cell-culture media, tissue, and swab.
Various viral RNA / DNA can be extracted, such as Adenovirus, Norovirus, Zika Virus, HCV, HIV, Yellow fiber virus, etc.
Application
  • Quantitative PCR (qPCR, qRT-PCR)
  • Pathogen detection
  • Poly-A selection
  • cDNA synthesis
  • Microarray
  • Northern blotting
Feature
  • High purity viral RNA / DNA extraction from various clinical samples
  • High purity viral RNA extraction without adding carrier RNA
  • Use of Glass fiber membrane column
  • Use a non-alcoholic primary washing buffer to remove impurities from the specimen.
  • Easy extraction other than Proteinase K without extra enzyme processing
  • Safe extraction without the use of organic solvents
QC Data

Comparison of Amplification Efficiency of Extracted Viral RNA/DNA (qRT-PCR / qPCR)

Components
Components
VL 30 ml
VW-1 60 ml
VW-2 (100% Ethanol) Empty bottle (provided)
100% Ethanol Not Provided
RNase free water 10 ml
Proteinase K (20mg/ml) 4 EA
Capsule Column 100 Preps
Quick Guide 1 EA
Know-How for Preparation
  1. How to prevent the cap of the tube from breaking during elution

    Please Inset 1.5 ㎖ tubes into centrifuge with their caps crossed to prevent them from breaking.
  2. It is recommended to use fresh sample for isolation.
  3. Please remove EtOH completely through centrifugation before elution step.
  4. Add dried enzyme to D.W(Buffer) and mix well. Please store it at -20°C.
  5. Please use the product according to its expiration date.
  6. Storage of VL at lower temperature may cause precipitation of salts due to SDS.
    In this case, please use it after thawing in the microwave/dry oven.
  7. It is recommended to use RNase free water after pre-heating (10 mins, at 50°C) during elution step for maximal recovery with the Kit.
    (Especially, high efficiency can be achieved in case of large DNA fragment.)
Troubleshooting
Trouble Check List
Low Yield DNA
01. Did you use fresh samples?

It is recommended to use fresh virus sample and avoid repeated, multiple freeze-thaw cycles to reduce the risk of viral DNA / RNA degradation. It should be stored in small working aliquots not to dissolve it more than 3 times.

02. Did you use Binding Buffer / VW-2 as 100% EtOH?

Lower percentages of Ethanol or use of other alcohol instead of EtOH may prevent efficient isolation of viral DNA/RNA. Please use 100% Ethanol only.

03. Where did you store the enzyme (Proteinase K)?

Ambient temperatures may harm the enzyme’s activity and stability in case of enzyme dissolved in D.W or enzyme with bu er added. It is recommended to be stored at -20°C for long-term storage.

04. How about incubation time after addition of Lysis Buffer?

Short incubation time leads to reduced recovery and it may decrease the yields.
Please follow incubation time speci ed for this protocol.

05. Did you add RNase free water directly to column membrane?

Make sure that you add RNase free water directly to the membrane during elution step.
Please incubate it for 1 min at RT after addition of RNase free water.

Nicked DNA
Degraded DNA
01. Have you thought about nuclease contamination?

Please check all plasticwares such as tips, microcentrifuge tubes and buffers for nuclease contamination before use.
Tips and microcentrifuge tubes should be autoclaved to avoid nuclease contamination.

Low Quality DNA
01. Did you dry Ethanol sufficiently after the washing step?

In case eluted viral DNA/RNA contains ethanol, it may cause problems for the next experiment.
It is recommended to remove Ethanol completely after washing step.

Work Flow
Preparation
  • 1. DIAKEY REALcheck Viral DNA/RNA Prep Kit is des igned to isolate viral DNA/RNA using 150 ㎕ of serum, plasma, cell-culture media and cell free body uids sample.
    (If the sample volume is less than 200 ㎕ , adjust the volume to 150 ㎕ with 1X PBS.)
  • 2. Although viral RNA/DNA is isolated regardless of use of 2-Mercaptoethanol (2-ME) on this kit, it is recommended to add 10 ㎕ of 2-ME (100%, optional) per 1 ㎖ of VL solution (at a ratio of 100:1)
    prior to use. Especially, high-quality RNA/DNA can be obtained through rapid inactivation of intracellular RNase and removal of potential PCR inhibitors when using 2-ME. In addition, higher detection sensitivity can be achieved through this treatment during Real-time PCR amplification.
  • 3. Prepare 100% Ethanol (not provided) into VW-2 bottle . Make it fresh.
Protocol
  • [Cell Lysis]
    1 : Add 150 ㎕ Sample to prepared 250 ㎕ VL and perform vortexing for 30 sec – 1 min.
    → Incubate the mixture for 10 mins at RT.
    * In case of Enveloped virus, please incubate the mixture after addition of 20 ㎕ Proteinase K solution (20 mg/ ㎖ ).
  • [Column Binding & Washing] 2 : Add Lysate (Step 1) to 350 ㎕ Ethanol (100%) and perform vortexing for 30 sec – 1 min.
    → Transfer it into capsule column and perform centrifugation at 13,000 rpm for 1 min.
    → Discard the flow-through solution
  • [Column Washing] 3 : Add 500 ㎕ VW-1 into capsule column and perform centrifugation at 13,000 rpm for 1min.
    → Discard the flow-through solution
    4 : Add 500 ㎕ VW-2 (100% Ethanol) into capsule column and perform centrifugation at 13,000 rpm for 1min.
    → Discard the flow-through solution
    5 : Perform centrifugation at 13,000 rpm for 1min to remove residual Washing Buffer (Ethanol)
    → Discard collection tube and place capsule column into new 1.5 ㎖ micro tube
  • [Viral DNA/RNA Elution] 6 : Add 50 ㎕ RNase free water into capsule column and incubate it for 1 min at RT
    → Perform centrifugation at 13,000 rpm for 1min and remove the column
    → Store at -20°C (viral DNA) & store at -70°C (viral RNA)
Order Info
Product Cat No. Size
DIAKEY® REALcheck Viral DNA/RNA Prep Kit DP015 50 Prep
DP0100 100 Prep