Introduction

The Kit is specially designed for simultaneous purification of genomic DNA and total RNA from formalin-fixed, paraffin-embedded (FFPE) tissue sections. The Purified DNA/RNA is used for RT-PCR and PCR detection.

Details

Specifications

Features	Specifications
Main FunctionsC	Co-isolation DNA and RNA from a single FFPE tissue sample
Applications	RT-PCR, cDNA synthesis, PCR and second-generation sequencing, etc.
Purification method	Mini spin column
Purification technology	Silica technology
Process method	Manual (centrifugation or vacuum)
Sample type	FFPE slice, FFPE embedded tissue
Sample amount	No more than six 10µm sections of 150mm2 surface area or three 20µm sections of 150mm2 surface area.

Principle

FFPE samples are incubated in an optimized lysis buffer, which results in the release of RNA and precipitation of DNA. After centrifugation, the RNA-containing supernatant and DNA-containing pellet are then processed separately to purify RNA and DNA. For RNA purification, transfer RNA Lysate to an adsorption column and RNA is adsorbed on the membrane, while protein is not adsorbed and is removed with filtration. After washing proteins and other impurities, RNA was finally eluted with low-salt buffer. For DNA purification, transfer DNA Lysate to an adsorption column and DNA is adsorbed on the membrane, while protein is not adsorbed and is removed with filtration. After washing proteins and other impurities, DNA was finally eluted with low-salt buffer.

Advantages

1.Use non-toxic dewaxing solution without contact with xylene
2.Obtain both DNA and RNA simultaneously from the same sample. Elute separately without affecting each other (Have the same steps and effects as top brand 80234, perfect substitute.)

Kit Contents

Contents	IVD5116
Purification Times	50 Preps
HiPure DNA Micro Column	50
HiPure RNA Mini Column I	50
2ml Collection Tubes	150
Proteinase K	50 mg
Protease Dissolve Buffer	5 ml
Buffer DPS	60 ml
Buffer FRL	15 ml
Buffer ATL	15 ml
Buffer RLC	15 ml
Buffer AL	15 ml
Buffer VHB	44 ml
Buffer RW2	25 ml
RNase Free Water	10 ml
Buffer AE	10 ml

Storage and Stability

Proteinase K should be stored at 2-8°C upon arrival. However, short-term storage (up to 12 weeks) at room temperature (15-25°C) does not affect their performance. The remaining kit components can be stored at room temperature (15-25°C) and are stable for at least 18 months under these conditions.

Experiment Data

The Kit is specially designed for simultaneous purification of genomic DNA and total RNA from formalin-fixed, paraffin-embedded (FFPE) tissue sections. The Purified DNA/RNA is used for RT-PCR and PCR detection.

Propargyl-PEG5-t-butyl ester has one of the functional groups protected by t-butyl group. The protection can be removed under acidic conditions. The propargyl group forms triazole linkages with azide-bearing compounds or biomolecules via copper catalyzed Click Chemistry. The PEG units help improving the water-solubility of the molecule in aqueous media. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.

Propargyl-PEG5-t-butyl ester has one of the functional groups protected by t-butyl group. The protection can be removed under acidic conditions. The propargyl group forms triazole linkages with azide-bearing compounds or biomolecules via copper catalyzed Click Chemistry. The PEG units help improving the water-solubility of the molecule in aqueous media. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.

• HiDi® stands for High Discrimination of mismatches at the 3’-terminus of primers in PCR. This myPOLS Biotec enzyme family is optimized for this feature and is the first choice for applications that rely on this property such as allele-specific PCR (asPCR) that is also termed allele-specific amplification (ASA).This polymerase is also available as a full-length Taq DNA polymerase with a nuclease domain, featuring 100% compatibility with hydrolysis probes (TaqMan® probes etc.).Benchmarking with products of competitors conducted by us and others show that the HiDi® DNA polymerase family is the first choice for highly selective PCRs, such as genotyping by allele-specific PCR, HLA genotyping, analysis of single CpG methylation sites or the detection of mutations in a high background of wild-type sequences. By using HiDi® DNA polymerase, less than 10 copies of a mutation can be detected in a background of >10.000 wild-type copies straight away without any other tedious assay optimization.Several independently conducted studies show that HiDi® DNA polymerase is ideally suited for use in asPCR in numerous research areas ranging from mutation detection to genome editing. (read more)
  For research use and further manufacturing.In case you are aiming to use our RUO products as components or for your development of e.g. an IVD medical device, please contact us.

HiDi is available as:

HiDi® DNA Polymerase (>>)
HiDi® Taq DNA Polymerase (>>)
HiDi® 2x PCR Master Mix (>>)
HiDi® Taq 2x PCR Master Mix (>>)

Casestudies:
HiDi® DNA Polymerase: Applications from mutation detection to genome editing (read more)

Example Primer Design

Matching vs. mismatching nucleotide is placed at the 3′-end of the primer for best discrimination results.

Example Results – There´s no accounting for taste

Cilantro: some people love it in their food, some hate it. Here we are detecting a genomic SNP (rs72921001) in HeLa genomic DNA. This SNP is reported to be close to a number of genes coding for olfactory receptors. (Reference: Eriksson N. et al. (2012), “A genetic variant near olfactory receptor genes influences cilantro preference.”)

Considering, that only the C-allele specific primer is extended and yielding in a specific amplicon, we can conclude a genetic predisposition in disliking cilantro, as this SNP is significantly associated with detecting a soapy taste to cilantro.  

Allele-specific PCRs were performed from 1 ng/µl of HeLa gDNA in the presence of a realtime dye, indicating the amplification of the C-allele specific primer only. The A-allele specific primer is discriminated, thus not amplified up to 50 cycles.

PCR products were subsequently analysed on a 2.5% agarose gel. Specific product is visualized by ethidium bromide staining at the amplicon length of 109 bp.

HiDi® stands for High Discrimination of mismatches at the 3’-terminus of primers in PCR. This myPOLS Biotec enzyme family is optimized for this feature and is the first choice for applications that rely on this property such as allele-specific PCR (asPCR) that is also termed allele-specific amplification (ASA).