DIAKEY® REALcheck Viral DNA/RNA Prep Kit
[For Column Type]
DIAKEY REALcheck Viral DNA/RNA Prep Kit is to extract viral DNA/RNA from various samples such as serum, plasma, cell-free body fluids, cell-culture media, tissue, swab, etc., and the kit can be safely used without using an organic solvent. Various viral DNA/RNA such as Adenovirus, Norovirus, Zika virus, HCV, HIV, Yellow fiber virus, Coronavirus, etc. can be extracted with high purity result.
- Quantitative PCR (qPCR, qRT-PCR)
- Pathogen detection
- Poly-A selection
- cDNA synthesis
- Northern blotting
- 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
Comparison of Amplification Efficiency of Extracted Viral RNA/DNA (qRT-PCR / qPCR)
|VW-2 (100% Ethanol)
||Empty bottle (provided)
|RNase free water
|Proteinase K (20mg/ml)
Know-How for Preparation
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.
- It is recommended to use fresh sample for isolation.
- Please remove EtOH completely through centrifugation before elution step.
- Add dried enzyme to D.W(Buffer) and mix well. Please store it at -20°C.
- Please use the product according to its expiration date.
- 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.
- 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.)
|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 buffer 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 specified 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.
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.
- 1. DIAKEY REALcheck Viral DNA/RNA Prep Kit is designed to isolate viral DNA/RNA using 150 ㎕
of serum, plasma, cell-culture media and cell free body fluids sample.
(If the sample volume is less than 150 ㎕ , 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.
|DIAKEY® REALcheck Viral DNA/RNA Prep Kit