direct dna/ rna/ protein...

USER GUIDE

Straightforward & Precise

Direct DNA/ RNA/ Protein Quantification

For Research Use Only

Not Intended for Any Animal or Human Diagnostic or Therapeutic Use

www.directquant.eu


2 www.directquant.eu

#DCQ100T DireCtQuant 100T ‐ DNA/RNA/protein solubilization reagent with temperature indicator #DCQ100W DireCtQuant 100W ‐ Colourless DNA/RNA/protein solubilization reagent #DCQ1100T DireCtQuant 1100T ‐ DNA/RNA/protein solubilization reagent for liquid samples with temperature indicator #DCQELM DireCtQuant ELM ‐ Electrophoresis loading media with temperature indicator #DCQDNAQ DireCtQuant DNAQ ‐ DNA quantification reagent #DCQSKIT DireCtQuant SKIT ‐ Selection kit of our new line of reagents



The DNA/RNA/protein solubilization reagents DireCtQuant 100T and 100W are

conceptually new reagents enabling quantitative analysis of a biological sample in a

very straightforward and precise way. The usage of these reagents overcomes the

traditional thinking that the building blocks of the biological systems (DNA, RNA and

proteins) should be separated prior to analysis. The complete solubilization of the

sample permits quantitative extraction and analysis of genomic, plasmid, plastid,

mitochondrial and viral DNA in the sample. RNA is also fully represented and

quantitative analysis of messenger, structural (such as ribosomal), transport,

microRNA, non‐coding RNA and viral RNA is achievable. This line of reagents provides a

one‐step, single reagent workflow which works by instant disruption of the structure

of the biological sample throw elimination of the non‐covalent interactions between

the biopolymers and bringing to solution its constituents. The solubilized DNA can be

directly analysed by PCR, quantitative (Real Time) PCR (qPCR) or quantified in solution.

The solubilized RNA can be analysed by reverse transcription coupled with quantitative

PCR (RT‐qPCR). The proteins can be analysed by Western blot. The instant disruption of

the structure of the cell and the biopolymers effectively blocks the endogenous

enzymatic activity of the sample, preserving its original composition and providing

exceptional storage capability. These reagents can be used with samples from

milligram to gram quantities of tissue or cultured cells (adherent or in suspension)

from a single cell to samples consisting up to million cells per millilitre of reagent. The

unique characteristic of DireCtQuant 100T and 100W is the possibility to perform an

extensive analysis of the DNA, RNA and protein levels from a single, minute sample.

DireCtQuant 1100T is a highly concentrated reagent, specifically designed for the

analysis of biological liquids containing a limited amount of cells or extracellular nucleic

acids. This includes, but it´s not limited to, serum, plasma, cerebrospinal fluid, amniotic



liquid. DireCtQuant 1100T provides the possibility of direct analysis and long‐term

sample storage capability.

DireCtQuant ELM is an electrophoresis loading media. Innovatively, following the

sample preparation procedures, this media changes the colour of the sample which

helps assure that the correct temperature in each procedure step has been reached. It

contains a novel tracking dye with a sharply defined migration front and no

fluorescence detection interference.

DireCtQuant DNAQ is a DNA quantification reagent which permits fast and accurate

quantification of DNA content in the sample. This reagent permits sample

normalization to be done in a unique way, based on the estimation of the genomic

DNA content. It is specifically designed to be compatible with the widely available

green fluorescence measuring equipment as fluorescence microplate readers,

microfluorimeters or qPCR platforms.

DireCtQuant SKIT is a selection kit of our new line of reagents: DCQ100T (9 ml),

DCQ100W (9 ml), DCQ1100T (0.9 ml), DCQELM (1.2 ml), DCQDNAQ (0.2ml).



TECHNOLOGY BEHIND

DireCtQuant 100T/100W/1100T are a result of a sophisticated manufacturing process.

The reagents are stable microemulsions at a precise equilibrium state. Upon contact

with the biological sample at high temperature the reagents effectively disrupt its

structure and instantly induce a long‐term suppression of the enzymatic activities.

Individual biopolymers are encased in a protective shell which guarantee long term

storage capability. Upon mixing with water an abrupt structural transition is induced,

which then permits analysis by enzyme mediated assays such as PCR. It is important

not to alter the composition of the extraction reagent and to use the recommended

sample to reagent ratio in order to preserve these qualities.

DireCtQuant 100T and 1100T contains an innovative temperature indicator which

permits rapid visual confirmation that the adequate temperature that is required for

the activation of the reagent is reached. The indicator is specifically selected not to

interfere with the polymerase activity in the PCR reaction and the fluorescence

excitation and detection in the region 470‐570 nm. It doubles as a colour guide during

the PCR setup, giving a pale red colour after addition of the sample to the PCR

reaction.

The same temperature sensing technology is used in our electrophoresis loading media

(DireCtQuant ELM) designed for direct analysis of protein content by Western blotting.

The achievement of the proper temperature for protein denaturalization and disulfide

bonds reduction and for gel loading is easily visually monitored.



CONTENTS

REAGENTS AND EQUIPMENT TO BE SUPPLIED BY USER 7

STORAGE 7

SAMPLE PREPARATION 8

Mammalian Tissue Samples Solubilization 8

Mammalian Adherent Cells Solubilization 10

Solubilization of Mammalian Cells Grown in Suspension Culture 12

Bacterial and Yeast Samples Solubilization 13

Plant Tissue Samples Solubilization 15

Liquid Samples Processing 16

SAMPLE ANALYSIS 17

qPCR or RT‐qPCR 17

Protein SDS‐PAGE 19

DNA Quantification 21

SAMPLE STORAGE 25

APPLICATION NOTE 26

Mouse Genotyping 26



REAGENTS AND EQUIPMENT TO BE SUPPLIED BY USER

● Dounce homogenizer/ Potter‐Elvehjem homogenizer/ Bead Beater

● Microcentrifuge

● Thermo block capable of maintaining 90°C, preferably with agitation

● Nucleases/proteases‐free water

STORAGE

DireCtQuant 100T/100W/1100T ‐ shipped at ambient temperature, upon arrival store

at room temperature up to 1 year.

DireCtQuant ELM ‐ shipped at ambient temperature, upon arrival store at ‐20°C up to

1 year. Once opened store at 4°C for up to 1 month.

DireCtQuant DNAQ ‐ shipped at ambient temperature, upon arrival store at ‐20°C up

to 1 year. Once opened store at 4°C for up to 1 month. Protect from unnecessary

exposure to light.



SAMPLE PREPARATION

Mammalian Tissue Samples Solubilization

1. Weigh the tissue and add 3.0 ml of DireCtQuant 100T/100W for every 100 mg of

tissue sample.

* NOTE: Both fresh and snap frozen in liquid nitrogen tissues can be used.

2. Homogenize completely with Dounce, Potter‐Elvehjem homogenizer or Bead Beater.

↕ CRITICAL: Complete homogenization of the tissue is critical. Usually 20 strokes of

Dounce homogenizer with tight clearance (approx. 50 µm) are sufficient. No particles

after homogenization should be visible by eye. Turbidity in case of lipid rich tissues

(such as brain, liver, etc.) is expected and normal.

↕ If you are solubilizing tissues rich in collagen, keratins or cartilage it will not be

completely solubilized and a pellet in step 5. will be observed. DireCtQuant 100T/100W

is not causing covalent bond breakage and because of this it can´t solubilize that kind

of extracellular matrix. In spite of this, DireCtQuant 100T/100W will effectively extract

all soluble components (DNA, RNA and not cross‐linked proteins) into the supernatant.

Homogenize as completely as possible with the appropriate method.

3. Transfer the lysate to an appropriate vial which can be securely closed, i.e vials with

screw cap. Use a vial with a volume similar to the volume of your sample in order to

minimise volume changes due to evaporation in the next step.

4. Heat at 90°C for 3 minutes with shaking at 750 rpm or manually inverting the vial

three to five times during the heat incubation.

! CAUTION: Use appropriate protection when handling hot vials.

* NOTE: DireCtQuant 100T only: the colour of the solution will turn orange upon reaching

the correct temperature.

5. Leave to cool down to room temperature and centrifuge at 5000 rcf for 1 minute.

* NOTE: With DireCtQuant 100T only the colour of the solution will turn back to its

original red colour.



6. If a pellet is observed, transfer the supernatant to a new vial.

7. Proceed with the analysis or store at ‐20°C (see sample storage).

* NOTE: With DireCtQuant 100T only upon freezing the colour of the solution will acquire

pink colour.



Mammalian Adherent Cells Solubilization

1. Completely aspirate the cell culture medium from plates.

2. Add minimum 200 µl of DireCtQuant 100T/100W per 1 cm2 of confluent cell layer in a

plate. Pipette several times until the whole cell layer is completely homogenized.

3. The optimal extraction ratio is 1000 cells/µl of DireCtQuant 100T/100W.

* NOTE: The maximum recommended number of cells per µl of DireCtQuant 100T/100W

is 2500 diploid human/rat/mouse cells. For some cell types, as aneuploid cell lines, the

number of cells should be reduced to avoid viscosity.

4. Collect the all of the homogenate (using cell scraper for example) and transfer the

lysate into a centrifuge vial.

5. Mix the cell lysate with a pipette (pipetting up and down three to five times is

usually sufficient) until the sample appears homogeneous.

* NOTE: If the lysate is too viscous and is difficult or not possible to pipette comfortably,

this means that the amount of cells is higher than the maximum recommended ratio of

2500 diploid human/rat/mouse cells per µl of DireCtQuant 100T/100W. In such a case add

50% more DireCtQuant 100T/100W to the homogenate.








! CAUTION: Use appropriate protection when handling hot tubes.

* NOTE: With DireCtQuant 100T only the colour of the solution will turn orange upon

reaching the correct temperature.

8. Leave to cool down to room temperature.

* NOTE: With DireCtQuant 100T only the colour of the solution will turn back to original

red.


* NOTE: With DireCtQuant 100T only upon freezing the colour of the solution will acquire

pink colour.



Solubilization of Mammalian Cells Grown in Suspension

Culture

1. Count the number of cells in the suspension culture.

2. Collect the cells by centrifugation and discard the supernatant completely.

3. Resuspend the pellet in DireCtQuant 100T/100W using the optimal ratio of 1x106

diploid human/rat/mouse cells per ml of DireCtQuant 100T/100W.

4. Mix the cell lysate with a pipette until it appears homogeneous. Pipetting up and

down three to five times is usually sufficient.







* NOTE: With DireCtQuant 100T only the colour of the solution will turn pale orange

upon reaching the correct temperature.



red.


* NOTE: With DireCtQuant 100T only upon freezing the colour of the solution will turn

pink.



Bacterial and Yeast Samples Solubilization

1. Collect the cells by centrifugation at 10000 rcf for 1 minute and discard all of the

media.

* NOTE: Bacteria and yeast posses a cell wall, which represent an extensive cross‐linked

structure which will be not solubilized, but independently of this the nucleic acids and

the soluble proteins will be effectively extracted.

2. Resuspend the pellet in DireCtQuant 100T/100W using the optimal ratio of 1x107

bacteria or 1x106 yeast cells per ml of DireCtQuant 100T/100W until appears

homogeneous.







* NOTE: With DireCtQuant 100T only the colour of the solution will turn pale orange upon




red.



6. Centrifuge at 10000 rcf for 1 minute and transfer the supernatant to a new vial.

Discard the pellet.


* NOTE: With DireCtQuant 100T only upon freezing the solution will acquire pink colour.



Plant Tissue Samples Solubilization

1. Weigh the tissue and add 3.0 ml of DireCtQuant 100T/100W for every 100 mg of tissue

sample.

* NOTE: Plant tissue contains a cell wall, which represent an extensive cross‐linked

structure which will not be solubilized, but independently of this the nucleic acids and the

soluble proteins will be effectively extracted in case mechanical disruption of the cell wall

is achieved. In case of very water‐rich tissue such as mature fruits reduce the amount of

DireCtQuant 100T/100W or consider using DireCtQuant 1100T for liquid samples. In the

latter case use 1/10 of the suggested volume of the reagent.

2. Homogenize completely with Bead Beater.

↕ CRITICAL: Complete homogenization of the material is critical. Optimize the time, size

and material of the beads to achieve complete homogenization. Alternatively freeze in

liquid nitrogen, grind to powder and add DireCtQuant 100T/100W.


screw cap. Use a vial with a volume similar to the volume of your in order to





* NOTE: With DireCtQuant 100T only the colour of the solution will turn pale orange upon


5. Leave to cool down to room temperature and centrifuge at 5000 rcf for 1 minute.

* NOTE: With DireCtQuant 100T only the colour of the solution will turn back to original red.

6. Transfer the supernatant to a fresh vial.


* NOTE: With DireCtQuant 100T only upon freezing the solution will acquire pink colour.



Liquid Samples Processing

DireCtQuant 1100T provides an unique opportunity to analyze the cell‐free

(circulating, exosome‐associated) nucleic acids without the purification steps usually

associated with significant nucleic acid loss. With a properly optimized assay, detection

of single molecule per µl of sample is possible.

1. Mix the sample (serum, plasma, cerebrospinal liquid) with one tenth of its volume of

DireCtQuant 1100T (e.g. mix 1 ml of plasma with 100 µl of DireCtQuant 1100T)

2. Use a vial which can be securely closed, i.e vials with screw cap. Use a vial with a

volume similar to the volume of your sample in order to minimise volume changes

due to evaporation in the next step.



! CAUTION: Use appropriate protection to when handling hot tubes.

* NOTE: The colour of the solution will turn orange upon reaching the correct temperature.


* NOTE: The colour of the solution will turn back to original red.


* NOTE: Upon freezing the colour of the solution will turn pink.



SAMPLE ANALYSIS

qPCR or RT‐qPCR

1. Use up to 1 µl of the sample solubilized with DireCtQuant 100T/100W/1100T for

reaction with a final volume of 20 µl (qPCR/RT‐qPCR).

First add the sample and the water to the reaction tube and mix well. Add the

enzyme/enzyme containing master mix as a last component. Undiluted reagent will

inhibit the polymerase activity. Don´t use more than 1 µl per 20 µl of final volume

PCR reaction!

The colour indicator in DireCtQuant 100T/1100T doubles as a colour guide and after

addition of the sample to PCR reaction it acquires pale pink colour. This does not

interfere with the excitation/detection of the fluorescence in the 430nm ‐ 560nm

range (this also improves signal to noise ratio). If you are using detection method not

within this spectral region it is necessary to use DireCtQuant 100W.

* NOTE: Suggested format for qPCR/RT‐qPCR analysis is 1000 cells per reaction. This

guarantees low variability and a confident detection of expression as low as single

gene/transcript copy per cell. Using a significantly higher number of cell per reaction will

likely inhibit the PCR reaction and does not represent any advantage in the detection of

low‐copy number targets.

Please consult DNA quantification section.

* NOTE: We strongly recommend the use of one‐step RT‐qPCR kits. Tested with Amethys One

Step qRT‐PCR MasterMix (Cambridge Bioscience). TaqMan® RNA‐to‐Ct™ 1‐Step Kit

(Applied Biosystems®), iTaq Universal One‐Step Kit (Bio‐Rad),

2. In the case that a two‐step RT‐qPCR is performed, use up to 1 µl of the lysate for the

first step (first strand cDNA synthesis) in final volume of 20 µl. The reverse

transcription product (cDNA) can be directly used in RT‐qPCR or stored at ‐20°C.

Usually up to 2 µl of cDNA is used for the second step reaction with total volume of

20 µl.



! CAUTION: Make sure that you are using exon‐exon spanning primer in your RT‐qPCR

assay as no separation of DNA and RNA is performed and they are both present in your

sample.



Protein SDS‐PAGE

1. Prepare the sample by mixing with:

DireCtQuant ELM (provided as 5X)

‐ Up to 31 µl of the sample extracted with DireCtQuant 100T/100W

‐ 8 µl of DireCtQuant ELM

‐ 1 µl of a reducing agent of your choice (2‐mercaptoethanol,

1M dithiothreitol or 0,5M TCEP), not provided.

* NOTE: 2‐Mercaptoethanol/β‐mercaptoethanol (2‐ME) is usually supplied as a 14M

solution (pure liquid) which will result in 360 mM final concentration if 1 µl is used for the

sample preparation.

Dithiothreitol (DTT) should be freshly prepared as a 1M solution in water (the resulting

final concentration will be 25 mM).

Ttris(2‐carboxyethyl)phosphine) (TCEP) should be prepared as a neutral pH solution. The

stock is stable at room temperature (the resulting final concentration will be 12,5mM).

This stable, non‐odor, non volatile, and irreversible reducing agent represents

significant advantage in handling, reproducibility and reduced toxicity.

* NOTE: Suggested format for Western blot is 10,000 ‐30,000 cells per 5 mm wide lane,

depending on the detection condition. Please use only non‐diluted DireCtQuant

100W/100T to adjust sample volumes. Don´t use water, this will interfere with the

migration of proteins and tracking dye characteristics.

2. Heat at 90°C for 3 minutes with shaking at 750 rpm.

3. Let the sample to cool down to room temperature and load on the gel.

* NOTE: The DireCtQuant ELM contains a temperature indicator which serves as tracking

dye as well. The colour of the sample is changing as follows:



- ORANGE during the incubation at 90°C indicating proper reduction/protein

denaturalization conditions.

- RED when room temperature is reached and sample is ready for loading and during the

electrophoresis.

- PINK when stored at ‐20°C.

Some lots of reducing reagents contain inpurities which can interfere will the sensitivity

of the temperature indicator. In this case you will not observe the clear colour

transitions but if proper temperature incubation is performed the sample preparation

will be successful.

* NOTE: The tracking dye front is RED and clearly defined compared with traditional

bromphenol blue‐containing tracking dyes. The dye migrates in front of the leading ion in

the Laemmli system and because of its red colour is easily distinguishable from the

commonly used blue stained protein markers. No residual binding to proteins is observed

as in some cases with bromphenol blue. The absorption spectrum is specifically selected

not to interfere with in‐gel or membrane fluorescence detection by infrared scanners.

The loading media is completely compatible with Laemmli system and MOPS/MES

neutral electrophoresis systems. In case BisTris‐gels are used in combination with MES

buffer the dye front is not as sharp and the colour can be yellow, but the electrophoresis

assay will be completed correctly in spite of this.

* NOTE: This system is not suitable for native electrophoresis. The DireCtQuant ELM

loading media should not be used with samples extracted in a different from

DireCtQuant 100T/100W/1100T buffers. Most probably this will result in aberrant or

absence of protein migration.



DNA Quantification

As the DNA amount per cell is a constant (with few exceptions) by estimation of DNA

concentration in the extracted sample it’s possible to estimate the original amount of

cells per volume of solubilized sample. The calculated number of cells per volume of

sample is possibly the best way to normalize between samples of the same experiment

and between samples across different experiments, etc. This workflow eliminates the

necessity of the measurement of expression of reference genes or housekeeping

proteins amount to normalise the RT‐qPCR and Western blot data, respectively.

As this method gives a very precise estimation of the total number of cells, it can also

be used as a cell viability assay (e.g. if the experimental treatment changes the number

of cells due to toxicity, division suppression, etc.). The result is to adjust all samples to

a common concentration (e.g. 1000 cells/µl) after the measurement, thus avoiding

usage of various dilution factors for each assay set up. Use the reagent that was

initially for the sample solubilization procedure.

The DNA concentration in a sample of tissue/cells solubilized with DireCtQuant

100T/100W can be measured using a method, based on the fluorescence emission of

proprietary dye complex. The presence of proteins, lipids and RNA in the lysates

prepared with DireCtQuant 100T/100W does not interfere with the sensitivity of the

DNA measurement.

It is fast (minutes) and adapted for use with a fluorescence plate reader, micro‐

fluorimeter or qPCR setup.

As a DNA standard solution a purified DNA sample prepared by the user or commercial

supplier should be used. You can use lambda DNA or linearized and purified plasmid

preparation.

! CAUTION: Do not use supercoiled plasmids as standards and make sure the standard

DNA is dissolved in pure nuclease free water without additives (TRIS, EDTA, etc.)



1. Prepare the working solution of DireCtQuant DNAQ by diluting it a hundred times

with nuclease free water (e.g. 10 µl of the stock solution with 990 µl of distilled

water).

* NOTE: DireCtQuant DNAQ is supplied in DMSO. Bring the stock solution to room

temperature in advance and homogenize well before use. Always prepare the

DireCtQuant DNAQ solution just before use, because the dye is stable for only a few

hours. Keep it protected from light.

2. Prepare standard curve dilution series from a DNA standard (not provided) using

DireCtQuant 100T/100W. Use the reagent used for the solubilisation of your sample.

* NOTE: The recommended range of the standard curve is 25‐1 ng/µl (equivalent approx

5000‐200 mammalian cells/µl).

3. Mix 20 µl of the sample or standards with 20 µl of the working solution of

DireCtQuant DNAQ (prepared in point 1.) in a tube or PCR plate. If a microplate

reader is used mix 100 µl sample plus 100 µl working DireCtQuant DNAQ solution per

96‐well plate. For any other plate formats, scale appropriately. Protect the samples

from long term exposure (more than minutes) to light. Mix well at any step.

4. Measure the fluorescence of the samples and the standards (Excitation ʎmax=495nm;

Emission ʎmax=535nm). Microfluorimeter, fluorescence plate reader with appropriate

filter set or SYBR / FAM channel of a Real‐Time PCR system can be used.

* NOTE: If Real‐Time PCR system is used set up the machine for single measurement at

20°C and look at the raw data for SYBR / FAM channel.

5. Create a standard curve using the data from standard DNA serial dilutions and

extrapolate the concentration of your sample.

* NOTE: Depending of the technology of the instrument used the resulting standard curve

could be straight line or polynomal function. Use the proper curve fitting method and

expect to achieve r2>0.995.



6. If a sample has a concentration higher than the most concentrated point of the

standard curve dilute the sample (from point 3) further, using the reagent used for

sample solubilization (DireCtQuant 100T/100W).

7. From the extrapolated DNA concentration of your sample you can now calculate the

number of cells per µl of DireCtQuant 100T/100W extract. Assume that a cell

contains a diploid genome. Human genome contains 7 pg of DNA, mouse genome ‐

6.5 pg and rat genome ‐ 6.9 pg of DNA. Consider the dilution during preparation of

the sample.

Example: Extrapolated concentration of a DNA sample is 6.9ng/µl.

If rat cells/tissue has been used (6.9 pg DNA/diploid cell)

Number of cells per µl of the extract = 6900 pg/µl/ 6.9 pg/cell = 1000 cells/µl.

8. Use this number to normalize the data of your experiments. For example, analyse

equal amount of cells per RT‐qPCR reaction which eliminates the need of reference

gene expression measurement, where expression can be instable depending of the

experimental conditions. The same data can be used for the normalization of sample

loading on Western blot analysis eliminating the necessity of detection of the

housekeeping gene, which also can be regulated in your particular experiment. This

workflow greatly simplifies the experimental work, provides substantial cost

reduction and significantly improves significantly the reliability of your analysis. For

normalization of the loading volumes always use the reagent used for the initial

solubilization of the sample. Don´t use water or another “buffer” which can interfere

with the structure of the reagent and its properties.



Fluorescence spectra of DNAQ bound to double stranded DNA

DNAQ is a proprietary dye formulation exhibiting a high selectivity to double stranded DNA.

Equal amount of RNA or protein in solution are exhibiting just around 15% and 1% increase in

the signal, respectively. This permits correct estimation of the concentration of DNA in the

presence of the other cellular constituents.



SAMPLE STORAGE

This system provides exceptional storage stability of biological samples once

solubilized. Unprecedented stability provides a greater flexibility in experimental

planning, archiving and field storage of biological samples. Our tests have shown no

detectable degradation of DNA (less than 10% variation, assessed by qPCR), RNA (less

than 10% variation, assessed by RT‐qPCR) or protein (densitometry comparison of

protein bands separated by gel electrophoresis) in samples (DireCtQuant

100T/100W/1100T lysates) stored, after the extraction is completed in undiluted

regent:

● at 37°C for a week;

● at room temperature for a week;

● at 4°C for a week;

● at ‐20°C for 3 months;

● at ‐80°C for at least 6 months;

● up to eight heat (90°C) and cool (RT) cycles.

● up to eight freeze and thaw cycles.



APPLICATION NOTE

Mouse Genotyping

1. Cut a small piece of mouse tail (1 mm from an adult animal or 2 mm from a newborn

animal) or ear punch and put it in a 1.5 ml tube.

2. For each sample to be processed add 200 µl DireCtQuant 100T/100W and 50 µg

Proteinase K (not provided).

3. Collect to the bottom of the tube by centrifugation.

! CAUTION: Be cautious that the whole tail piece is covered by the solution.

4. Incubate samples at 55°C with gentle shaking. Usually it takes 1‐2 hours (newborn) or

2‐4 hours (adult) until the piece of tissue is completely digested. Alternatively,

incubate the tubes overnight at 37°C.

5. After the tissue is completely digested place the tubes at 90°C for 3 minutes in order

to inactivate the Proteinase K.


6. Proceed with the analysis or store at ‐20°C.

7. For PCR‐based genotyping analysis use directly up to 1 µl of the sample per 20 µl

reaction.

* NOTE: Please note that after the solubilization of the tail in the presence of Proteinase K

the lysate is not suitable for any other application except PCR‐based genotyping analysis.



General statement

Handle our products in accordance with safe laboratory practices:

wear suitable protective gloves, eyewear and clothing.

In case of any doubt please feel free to contact DireCtQuant team at:

[email protected]

Last version: V6

direct dna/ rna/ protein...

Documents