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USER GUIDE

Straightforward & Precise

Direct DNA/ RNA/ Protein Quantification

For Research Use Only Not Intended for any Animal or Human Diagnostic or Therapeutic Use

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#DCQ100T DireCtQuant 100T ‐ DNA/RNA/protein solubilization reagent with temperature indicator #DCQ100ST DireCtQuant 100ST ‐ DNA/RNA/protein solubilization reagent with stabilized 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, 100ST 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 through 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, effectively 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, 100ST 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: DCQ100ST (4.5 ml),

DCQ100W (4.5 ml), DCQ1100T (0.5 ml), DCQELM (0.6 ml), DCQDNAQ (0.1ml).



TECHNOLOGY BEHIND

DireCtQuant 100T/100ST/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 temperatures 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, 100ST and 1100T contain 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 protein 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 ● Micro centrifuge ● Thermo block capable of maintaining 90°C, preferably with agitation ● Nucleases/proteases‐free water

STORAGE DireCtQuant 100T/100ST/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/100ST/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/100ST/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/100ST/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 and 100ST 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 and 100ST 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 and 100ST 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 a minimum 200 µl of DireCtQuant 100T/100ST/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/100ST/100W. * NOTE: The maximum recommended number of cells per µl of DireCtQuant 100T/100ST/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/100ST/100W. In such a case add 50% more DireCtQuant 100T/100ST/100W to the homogenate.

6. 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 in order to minimise volume changes due to evaporation in the next step.




! CAUTION: Use appropriate protection when handling hot tubes.

* NOTE: With DireCtQuant 100T and 100ST 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 and 100ST only the colour of the solution will turn back

to original red.


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/100ST/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.

5. 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.


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

* NOTE: With DireCtQuant 100T and 100ST only the colour of the solution will turn pale orange upon reaching the correct temperature.

7. Leave to cool down to room temperature. * NOTE: With DireCtQuant 100T and 100ST only the colour of the solution will turn back to original red.


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/100ST/100W using the optimal ratio of 1x107 bacteria or 1x106 yeast cells per ml of DireCtQuant 100T/100ST/100W until appears homogeneous.

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.



* NOTE: With DireCtQuant 100T and 100ST only the colour of the solution will turn pale orange upon reaching the correct temperature.

5. Leave to cool down to room temperature. * NOTE: With DireCtQuant 100T and 100ST only the colour of the solution will turn back to original red.



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

7. Proceed with the analysis or store at ‐20°C (see sample storage). * NOTE: With DireCtQuant 100T and 100ST 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/100ST/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 consider the usage of DireCtQuant 1100T for liquid samples. In the latter case use 1/10 of the suggested volume of the reagent, e.g.:

Sample weight #DCQ100T #DCQ1100T 10 mg 0.3 ml 0.03 ml 100 mg 3 ml 0.3 ml 300 mg 9 ml 0.9 ml 600 mg 18 ml 1.8 ml

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/100ST/100W.

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 in order to minimise volume changes due to evaporation in the next step.


! CAUTION: Use appropriate protection when handling hot tubes. * NOTE: With DireCtQuant 100T and 100ST only the colour of the solution will turn pale 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 and 100ST only the colour of the solution will turn back to original red.

6. Transfer the supernatant to a fresh vial. 7. Proceed with the analysis or store at ‐20°C (see sample storage).

* NOTE: With DireCtQuant 100T and 100ST 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.



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

4. Leave to cool down to room temperature. * NOTE: The colour of the solution will turn back to original red.

5. Proceed with the analysis or store at ‐20°C (see sample storage). * 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/100ST/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/100ST/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/100ST/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).

Tris(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/100ST/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 impurities 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/100ST/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/100ST/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/100ST/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/100ST/100W).

7. From the extrapolated DNA concentration of your sample you can now calculate the number of cells per µl of DireCtQuant 100T/100ST/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/100ST/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/100ST/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.

! CAUTION: Use appropriate protection when handling hot tubes.

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]

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