Reagents for purification and isolation of nucleic acids synthol. Set of reagents for isolation of genomic DNA "DNA-Extran"

The rapid microcolumn DNA extraction method is designed for the extraction of total DNA from blood, plasma, saliva, urine, cell cultures and any cell pellets in buffer. The high purity of the isolated DNA allows it to be used for all types of analysis.

isolation time ranges from 30 to 45 minutes depending on the number of samples;

• effective binding of DNA to the column membrane allows you to obtain the highest yield of DNA from the sample;
• a high degree of purification is achieved thanks to the optimized composition of the components of the lysing and washing solutions;
• significantly reduced fragmentation and loss of DNA during washing compared to other sorbent extraction methods;
• it is possible to concentrate DNA by reducing the volume of the elution solution;
• DNA extraction on columns significantly reduces the consumption of additional plastic;
• maximum sample volume - 200 µl;
• the kit contains Proteinase K;
• the purity of the isolated DNA is 1.8-1.9 according to the ratio A260/A280;
• the amount of DNA extracted depends on the type and quantity of sample. The DNA yield from 200 µl of blood averages 5-6 µg.

Set of reagents for isolation of genomic DNA "DNA-Extran"

Using kits for the isolation of genomic DNA of the DNA-Extran series, you can isolate DNA from a variety of biological material: blood, cultures of bacterial and animal cells, fresh, frozen or dried tissues of animals and plants.

• complete DNA extraction from cells, minimizing DNA loss and fragmentation during purification;
• the isolated DNA has high level purity (ratio A260/A280 = 1.8-1.9) and suitable for PCR, restriction digestion, hybridization and other studies;
• the kits do not contain potentially hazardous components such as phenol and chloroform and do not require high flow rate plastic and do not create toxic waste.

Set of reagents for DNA extraction on magnetic particles "M-Sorb-Tube"

Adapted for the isolation of mycobacterial DNA from sputum, bronchial washings, cerebrospinal fluid, exudate, punctate, biopsy, urine, genital tract discharge, and cell cultures. Pre-processing of clinical material is carried out in accordance with standard procedures for sample preparation for microbiological studies (Order No. 109 of the Ministry of Health of the Russian Federation of March 21, 2003 “On improving anti-tuberculosis measures in the Russian Federation”, Appendix 11 “Instructions for unified methods microbiological research in the detection, diagnosis and treatment of tuberculosis"). To inactivate clinical material, we recommend using the inactivating solution A that we have developed.

Solution A kills mycobacteria within 12 hours and disinfects clinical material, which can be used for further analysis (DNA extraction, PCR, etc.). Solution A is adapted specifically for the treatment of sputum and completely replaces the standard procedure using NaOH-NALC solution and has exceptional mucolysing properties. Inactivation Solution A increases DNA yield compared to standard NaOH-NALC sample preparation.

The isolation technique includes: DNA lysis with guanidine isothiocyanate, DNA sorption on magnetic particles, DNA precipitation by centrifugation, washing stages and DNA elution. Can be used to isolate DNA from other microorganisms.

the use of silica gel-coated magnetic particles as a sorbent is a more technologically advanced and convenient format compared to other sorbents and allows for maximum standardization and automation of the DNA extraction technique;

An internal positive control (IPC) is added to each test sample; the presence/absence of RT-PCR inhibitors and the efficiency of DNA extraction are determined by the IPC amplification reaction.

Reagent kits for DNA extraction from food products and food raw materials

The “Sorb-GMO-A” and “Sorb-GMO-B” reagent kits are designed specifically for DNA extraction from plant materials, food products and feed. Both kits use silicon sorbent for DNA purification. “Sorb-GMO-A” contains guanidine chloride as a lysing agent, “Sorb-GMO-B” is an ionic detergent CTAB, which provide maximum DNA yield from plant components. Distinctive Features The advantages of the Sorb-GMO-A kit are the speed of DNA extraction and the absence of chloroform, which makes working with the kit safer.

Set of reagents for DNA extraction from water samples (on magnetic particles) "M-Sorb-Leg"

Designed for the isolation of Legionella DNA from water bodies environment(cooling towers, swimming pools, water parks, hot and cold water supply systems). The minimum isolated concentration of Legionella is 100 cells per 0.5 liter of sample.

The “M-Sorb-Leg” kit is produced in two modifications: “M-Sorb-Leg1000” for water samples with a volume of 1-1000 ml and “M-Sorb-Leg1” for water samples with a volume of up to 1 ml. The M-Sorb-Leg1000 set provides water filtration using a polycarbonate filter.

the M-Sorb-Leg reagent set allows you to get rid of PCR inhibitors present in heavily contaminated water samples;

• The DNA isolation technique is based on the sorption of DNA on silica gel-coated magnetic particles followed by precipitation with a precipitating reagent. Combines the advantages of sorption methods and the total precipitation method;
• An internal positive control (IPC) is added to each test sample; the presence/absence of RT-PCR inhibitors is determined by the IPC amplification reaction.

Set of reagents for DNA extraction from environmental objects (on magnetic particles) "M-Sorb-OOM"

The M-Sorb-OOM reagent set is intended for the isolation of DNA from environmental objects (soil, water, animal corpses, etc.) suspected of being infected with especially dangerous microorganisms (DOM), in order to prepare them for subsequent analysis by RT-PCR . The isolation procedure is similar to that described for the M-Sorb-Leg kit.

Set of reagents for RNA isolation "RNA-Extran"

The kit is intended for isolating RNA from blood, tissue fragments, and cell cultures. The RNA obtained using the RNA-Extran kit can be used both for RT-PCR and for hybridization analysis, in vitro translation, and cloning.

The principle of RNA isolation is based on acidic phenolic extraction according to Khomchinsky, in which only RNA remains in the aqueous phase, and DNA in complex with proteins passes into the organic phase. Guanidine thiocyanate is used as an isolating and denaturing agent for cellular nucleases.

allows you to obtain highly purified RNA, free from DNA impurities;

provides complete extraction of RNA from whole blood, tissue fragments and cell cultures;

allows you to obtain undamaged RNA;

• RNA isolation time - 1 hour.

Catalogue number	Name	number of reactions
EX-509	Set of reagents “DNA-Extran-1” for the isolation of genomic DNA from whole blood	100
EX-511	Set of reagents “DNA-Extran-2” for DNA extraction from animal and human tissues	100
EX-512	Set of reagents “DNA-Extran-3” for DNA extraction from bacterial cultures	100
EX-513	Set of reagents “DNA-Extran-4” for DNA extraction from plant tissues	100
EX-514	Set of reagents “K-Sorb” for isolation of total DNA on columns (from blood, saliva, urine, cell cultures, scrapings of epithelial cells)	100
EX-515	Set of reagents "RNA-Extran" for isolating RNA from blood, tissues, cell cultures	50
OM-505	Set of reagents “M-Sorb-Tub” for DNA extraction from clinical samples and cell cultures (on magnetic particles)	50 or 100
GM-502-50	“SORB-GMO-A” (guanidine + sorbent) Set of reagents for DNA extraction from plant materials and food products	50
GM-503-50	“SORB-GMO-B” (CTAB+sorbent) Set of reagents for DNA extraction from plant materials and food products	50
OM-506	Set of reagents “M-Sorb-Leg1000” for the isolation of Legionella DNA from water samples up to 1000 ml (on magnetic particles, filters are supplied separately)	50 or 100
OM-507	Set of reagents “M-Sorb-Leg1” for the isolation of Legionella DNA from water samples up to 1 ml in volume (on magnetic particles)	50 or 100
OOM-502	Set of reagents “M-sorb-OOM” for DNA extraction from environmental objects (on magnetic particles)	50 or 100

Ordering information

Name	Volume	Production	Method	Cat.No.
“GMO-MagnoSorb” (guanidine + magnetic sorbent) Set of reagents for DNA extraction from plant materials and food products	50 allocations	Synthol	Real-time PCR	GM-505-50
“SORB-GMO-A” (guanidine + sorbent) Set of reagents for DNA extraction from plant materials and food products	50	Synthol	Real-time PCR	GM-502-50-50
“SORB-GMO-B” (CTAB+sorbent) Set of reagents for DNA extraction from plant materials and food products	50	Synthol	Real-time PCR	GM-503-50-50
Set of reagents “Amplitub-RV” kit No. 1 (“M-Sorb-Tub“) for the isolation of mycobacterial DNA from clinical samples and culture cells (on magnetic particles)	50	Synthol	Real-time PCR	OM-505
Set of reagents “DNA-Extran-1” for the isolation of genomic DNA from whole blood	100	Synthol	Real-time PCR	EX-509-100
Set of reagents “DNA-Extran-2” for DNA extraction from animal and human tissues	100	Synthol	Real-time PCR	EX-511
Set of reagents “DNA-Extran-3” for DNA extraction from bacterial cultures	100	Synthol	Real-time PCR	EX-512-100
Set of reagents “DNA-Extran-3” for DNA extraction from plant tissues	100	Synthol	Real-time PCR	EX-513-100
Set of reagents “K-Sorb” for isolation of total DNA on columns (from blood, saliva, urine, cell cultures, scrapings of epithelial cells)	100	Synthol	Real-time PCR	EX-514-100
	48 reactions	Synthol	Real-time PCR	GM-443-48
Set of reagents “Soya / 35S+FMV / NOS screening”	50 reactions	Synthol	Real-time PCR	GM-416-50

Lysis solution 30 cm 3,

Washing solution 1 30 cm 3,

Concentrate for cleaning solution 2 20 cm 3,

Sorbent suspension 2 cm 3,

DNA elution buffer 4 cm 3 .

Operating procedure:

Prepare cleaning solution 2 by mixing 20 cm 3 of the concentrate from the kit, 80 cm 3 of distilled water and 100 cm 3 of 96% ethanol in a separate bottle. Store the solution at room temperature in a tightly screwed bottle.

Check the condition of the sorbent - when settling, it should occupy approximately half the volume of the suspension.

Into a tightly closed 1.5 cm 3 polypropylene tube (preferably with a screw cap), add 100 µl of a pre-prepared test sample, negative or positive sample, add 300 µl of lysis solution (to each sample with a separate tip) and mix thoroughly by pipetting 3- 10 times.

Add 15 - 20 µl of resuspended sorbent, mix well by vortex, place in a rack for 5-7 minutes for complete sedimentation of the sorbent.

Sediment the sorbent in a microcentrifuge for 30 seconds at 3-8 thousand rpm. Take the supernatant from each tube using a separate tip (it is convenient to use a vacuum suction).

Add 300 µl of washing solution 1, mix by vortex until the sorbent is completely resuspended (if the sorbent breaks down badly, break it up with a pipette), sediment in a centrifuge at 4-8 thousand rpm for 30 seconds. Collect the supernatant from each tube using a separate tip.

Add 800 μl of washing solution 2, vortex until the sorbent is completely resuspended, sediment in a microcentrifuge at 6-10 thousand rpm for 30 seconds, and collect the supernatant.

Repeat step 6, carefully select the supernatant, dry the sorbent sediment in a thermostat at 65 °C for 5 minutes.

Resuspend the sorbent in 30-40 μl of elution buffer, place in a thermostat at 65°C for 5 minutes, periodically shaking by vortex.

Sediment in a microcentrifuge at maximum speed for 1 minute.

The sample is ready for PCR; the supernatant contains purified DNA.

As a negative control during the DNA extraction step, it is necessary to use saline or deionized water.

The efficiency of DNA extraction using the DNA-sorb-PCR kit is 30 – 60%.

Clinical material	Plasma, serum	Scraping, sperm, pharyngeal washes, urine	Biopsy, blood, feces
Number of pipettings
Sorbent volume
Sorbent deposition rate	8 thousand rpm	6 thousand rpm	3-4 thousand rpm
Eluent volume
DNA extraction efficiency

5.2. Stage 2. Setting up PCR composition of the kit for PCR amplification:

5x reaction buffer. 1000 µl (viscous, transparent blue solution)

Deionized water.2000 µl

Mixture of nucleotides.500 µl

Taq polymerase.100 µl

Primer mixture.500 µl

Wax for PCR.2000 µl

Positive control: 100 µl

Negative control 100 µl

Mineral oil 4000 µl

The kit is stored at minus 20°C for a year.

"DNA-sorb-S-M" ® AmpliSens FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, Only for research Russian Federation, 111123, and other non-medical purposes, Moscow, ... "

INSTRUCTIONS

on the use of the reagent kit

for DNA extraction from biological material

"DNA-sorb-S-M"

AmpliSense

FBUN Central Research Institute of Epidemiology

Rospotrebnadzor,

For research purposes only

Russian Federation, 111123,

and other non-medical purposes

Moscow city, Novogireevskaya street, building 3A

LIST OF ABBREVIATIONS

PURPOSE

PRINCIPLE OF THE METHOD

FORMS OF COMPLETING

EXTRACTION OF DNA FROM BIOLOGICAL MATERIAL FROM ANIMALS................ 8

ANIMAL FEED OR PLANT RAW MATERIALS

SYMBOLS USED IN PRINTED PRODUCTS

Form 1: REF K1-6-50-Mod / VER 12/27/16 / page 2 of 15

LIST OF ABBREVIATIONS

In this manual, the following abbreviations and symbols are used:

DNA – deoxyribonucleic acid NA – nucleic acids OK – negative extraction control NCO – negative control sample PC – positive extraction control PKO – positive control sample PCR – polymerase chain reaction

– Federal state-financed organization Sciences

FBUN Central Research Institute

"Central Research Institute of Epidemiology and Epidemiology" Federal service for supervision in the field of Rospotrebnadzor for the protection of consumer rights and human well-being

PURPOSE

The DNA-sorb-S-M reagent kit is intended for DNA extraction from biological material from animals: tissue (biopsy (skin and mucous membranes of the genitourinary system, gastrointestinal tract, bronchi), surgical and autopsy) material; and DNA extraction from food, biological additives, animal feed or plant materials for subsequent research using polymerase chain reaction (PCR).

DNA extraction is a preanalytical procedure of the PCR method.

Volume of the test sample for extraction: 100 µl (samples with a volume of 10 to 100 µl or weighing from 10 to 100 mg are allowed)1.

ATTENTION! For information about the collection procedure, conditions for transportation and storage of the test material, the need and procedure for its preparation for DNA extraction, as well as information about interfering substances and restrictions associated with the sample, see the instructions for the set of reagents used for amplification.

PRINCIPLE OF THE METHOD

The test sample2 is treated with a lysis solution containing proteinase K, resulting in the destruction of cell membranes and the release of NK and cellular components. Dissolved NCs bind to sorbent particles, while other components of the lysed test material remain in solution and are removed during sorbent precipitation. Depending on the material being tested, see the instructions for the amplification reagent kit used.

Some types of biological material require a sample preparation step. Cm.

instructions for the reagent kit used for amplification.

Form 1: REF K1-6-50-Mod / VER 12/27/16 / page 3 of 15 by centrifugation and subsequent washing of the sorbent. When an elution buffer is added to the sorbent, the NC passes from the silica surface into the solution, which is separated from the sorbent particles by centrifugation. As a result of this procedure, a highly purified NA preparation is obtained, free from amplification reaction inhibitors, which ensures high analytical sensitivity of the PCR study.

FORMS OF COMPLETING

The reagent kit is available in 2 configurations:

Form 1 includes a set of reagents “DNA-sorb-S-M” option 50.

Form 2 includes a set of reagents “DNA-sorb-S-M” version 100.

PRECAUTIONS AND DISPOSAL INFORMATION

When studying biological material from animals, work must be carried out in accordance with the rules of the Ministry of Agriculture and Petroleum of the Russian Federation of January 27, 1997 No. 13-7-2/840 “Rules for carrying out work in diagnostic laboratories using the polymerase chain reaction method. Basic provisions”, approved by the Department of Veterinary Medicine.

When researching food products, biological additives, animal feed or plant materials, the work must be carried out in compliance with the requirements methodological instructions MU 1.3.2569-09 “Organization of the work of laboratories using nucleic acid amplification methods when working with material containing microorganisms of pathogenicity groups I–IV” and GOST R 53214-2008 “Food products. Analysis methods for genetic detection modified organisms and products obtained from them.

General requirements and definitions."

When working, you must always comply with the following requirements:

– The laboratory process should be unidirectional. The analysis is carried out in separate rooms (zones). Work should begin in the Extraction Zone and continue in the Amplification and Detection Zone. Do not return samples, equipment, or reagents to the area in which the previous process step was performed.

– Unused reagents, expired reagents, as well as Form 1: REF K1-6-50-Mod / VER 12/27/16 / page 4 of 15 used reagents, packaging3, biological material, including materials, instruments and items contaminated biological material, should be disposed of in accordance with the requirements of SanPiN 2.1.7.2790-10 “Sanitary and epidemiological requirements for the management of medical waste.”

– Use and change disposable tips for automatic filter pipettes with each operation4. Disposable plastic utensils must be disposed of in a special container containing a disinfectant that can be used for disinfection medical waste.

– Dishes (mortars and pestles) and metal instruments (scalpels, scissors, tweezers, blender attachments, etc.) used for sample preparation are kept in a disinfectant solution (for example, 0.2% solution of sodium salt of dichloroisocyanuric acid) for one hour, washed with tap water with surfactant detergents and, after washing in running and deionized water, dried in a dry-heat oven for 4 hours at a temperature of 180 C.

– The reagent kit is intended for one-time use to carry out the study of the specified number of samples (see section “Composition”).

– The reagent kit is ready for use according to these instructions.

Use the reagent kit strictly for its intended purpose.

– Do not use the reagent kit if the inner packaging is damaged or appearance The reagent does not match the description.

– Do not use the reagent kit if the transportation and storage conditions according to the instructions are not followed.

Unused reagents, expired reagents, used reagents, packaging belong to the hazard class of medical waste G.

Disposable tips without a filter are used to remove the supernatant during the extraction process using a vacuum aspirator.

Form 1: REF K1-6-50-Mod / VER 12/27/16 / page 5 of 15

– Do not use the reagent kit after the expiration date.

– Use disposable powder-free gloves, laboratory coats, and eye protection when working with samples and reagents. Wash your hands thoroughly after finishing work. All operations are carried out only with gloves to avoid contact with the human body.

– Avoid inhalation of vapors, contact with skin, eyes and mucous membranes, do not swallow. In case of contact, immediately rinse the affected area with water and, if necessary, seek medical attention.

– Reagent safety data sheets (SDS – safety data sheet) are available upon request.

Assessment of probable events that may result in negative consequences for the human body:

When used as intended and the above precautions are followed, contact with the human body is excluded.

At emergency situations the following is possible:

– irritation of the mucous membrane of the eyes in sensitive persons,

– skin irritation in sensitive persons,

- allergic reaction,

– harm if inhaled,

- Harmful if taken orally.

Specific effects of the reagent kit on the human body:

– There is no carcinogenic effect.

– There is no mutagenic effect.

– No reproductive toxicity.

ADDITIONAL MATERIALS AND EQUIPMENT

(indicating manufacturers/suppliers):

1. 1.5 and 5.0 mL disposable polypropylene screw-on or tight-seal tubes (e.g., Axygen, Inc.

("Exigen, Inc"), USA, or similar).

2. Disposable tips for variable volume pipettes with a filter up to 200 and up to 1000 µl (for example, Axygen, Inc., USA, or similar).

3. Disposable tips for variable volume pipettes up to 200 µl (for example, Axygen, Inc., USA, or similar).

Form 1: REF K1-6-50-Mod / VER 12/27/16 / page 6 of 15

4. Racks for 1.5 ml tubes (e.g. Axygen, Inc., USA, or similar).

5. Laminar flow hood, biological safety class II type A (for example, “BAVp-01-“Laminar-S”-1,2”, JSC “Laminar Systems”, Russia, or similar).

6. Thermostat for Eppendorf tubes from 25 to 100 °C (for example, SIA Biosan, Latvia, or similar).

7. Thermostat-shaker for Eppendorf tubes from 25 to 100 °C (for example, TS-100, SIA Biosan, Latvia, or similar).

8. Microcentrifuge for Eppendorf tubes with maximum speed centrifugation of at least 12 thousand g (for example, MiniSpin, Eppendorf (Eppendorf Manufacturing Corporation), Manufacturing Corporation Germany, or similar).

9. Vortex (for example, SIA Biosan, Latvia, or similar).

10. Medical vacuum aspirator with a trap flask for removing supernatant liquid (for example, “OM-1”, LLC “Utes”, Russia, or similar).

11.Automatic variable volume dispensers (for example, Biohit LLC, Russia, or similar).

12. Refrigerator from 2 to 8 °C with freezer from minus 24 to minus 16 C.

13.Separate robe, hats, shoes and disposable gloves according to MU 1.3.2569-09.

14.Disposable plastic containers for disposal and inactivation of materials.

–  –  –

EXTRACTION OF DNA FROM BIOLOGICAL MATERIAL FROM ANIMALS

2. Select the required number of 1.5 ml disposable polypropylene tubes with screw or tight-fitting caps (including negative and positive extraction controls, if provided for the PCR study).

When storing the lysis reagent buffer and wash solution 1 at a temperature of 2 to 8 C, the formation of a precipitate in the form of crystals is possible.

Form 1: REF K1-6-50-Mod / VER 12/27/16 / page 8 of 15

3. Add 10 µl of BKO6 to each tube (if it is provided for PCR research). Add 400 µl of lysis reagent buffer and 17 µl of lysis reagent to the tubes.

4. Add 100 µl of the test samples6 to the prepared tubes, using a separate tip with a filter for each sample.

ATTENTION! 400 µl of buffer for lysis reagent and 17 µl of lysis reagent can be added to a tube with the required amount of the test sample and 10 µl of BKO7 can be added there (if it is provided for PCR research), using separate tips with a filter.

5. Add 100 μl of NCO to the negative control (NC) extraction tube, add 90 μl of NCO and 10 μl of PCO to the positive control (PC) extraction tube (if extraction controls are provided for PCR studies).6

6. Close the lids tightly, mix thoroughly and vortex the drops.

Place the tubes in a thermostat with a temperature of 64 °C for 1 hour, periodically mixing by vortex (5 times every 10–12 minutes). Incubation is allowed for 12 hours at a temperature of 60 °C.

7. Pellet undissolved sample particles by centrifugation for 5 minutes at 10 thousand g (for example, 12 thousand rpm for a MiniSpin microcentrifuge, Eppendorf Manufacturing Corporation).

8. Take the supernatant liquid in a volume of 200–350 µl very carefully (avoiding suspended particles and fat droplets) using separate tips with filters and transfer to new tubes. Sediment the drops by vortex.

9. Resuspend the universal sorbent, stirring vigorously using a vortex. Add 25 µl of resuspended universal sorbent to each tube with a separate tip, close the lids tightly.

Mix by vortex, leave in a rack for 10 minutes, stirring every 2 minutes.

It is possible to change the volume of test and control samples; see the instructions for the used set of reagents for amplification.

Form 1: REF K1-6-50-Mod / VER 12/27/16 / page 9 of 15

18.Repeat the washing procedure following paragraphs. 15–16, remove the supernatant completely.

19. Place the test tubes with open lids in a thermostat at 64 °C for 5–10 minutes to dry the universal sorbent.

20.Add 50–100 μl of elution buffer B into the tubes (see instructions for the amplification reagent kit used).

Mix by vortex until the sorbent is completely resuspended. Place in a thermostat at 64 °C for 5–10 minutes, stirring periodically (once per minute) using a vortex.

Purified DNA can be stored at a temperature of 2 to 8 °C for a week, at a temperature of minus 24 to minus 16 °C for 6 months, and at a temperature not exceeding minus 68 °C for a year. To do this, it is necessary, without capturing the sorbent, to transfer the supernatant liquid into a new test tube.

Form 1: REF K1-6-50-Mod / VER 12/27/16 / page 10 of 15

EXTRACTION OF DNA FROM FOOD, BIOLOGICAL ADDITIVES,

ANIMAL FEED OR PLANT RAW MATERIALS

ATTENTION! For the procedure for preparing biological material for DNA extraction and the volume of the test sample, see the instructions for the amplification reagent kit used.

1. Warm the lysis reagent buffer and wash solution 1, if they were stored at a temperature of 2 to 8 °C, at a temperature of 64 °C until the crystals are completely dissolved.

2. Place 1.5 ml tubes with pre-processed test samples in a rack (sample volume/quantity, see instructions for the amplification reagent kit used).

3. Prepare a 1.5 mL disposable polypropylene tube with a screw or tight-fitting cap for the negative extraction control (EC). Add 100 µl of OKO to the test tube.

4. Add 400 μl of buffer for the lysis reagent and 17 μl of the lysis reagent to the test tubes with the test samples and TC.

5. Close the lids tightly, mix thoroughly and vortex the drops.

Place the tubes in a thermostat with a temperature of 64 °C for 1 hour, periodically mixing by vortex (5 times every 10–12 min), or use a shaker thermostat.

6. Pellet undissolved sample particles by centrifugation for 5 minutes at 10 thousand g (for example, 12 thousand rpm for a MiniSpin microcentrifuge, Eppendorf Manufacturing Corporation).

7. Select the required number of new disposable 1.5 ml polypropylene tubes with screw or tight-fitting caps.

8. Resuspend the universal sorbent, stirring vigorously using a vortex. Add 25 µl of resuspended universal sorbent to each tube with a separate tip, close the lids tightly.

9. From the tubes with lysed samples, take the supernatant liquid in a volume of 200–350 µl very carefully (avoiding the ingress of suspended particles and drops of fat) using separate tips with filters and transfer to tubes with a sorbent. Mix by vortex, leave in a rack for 10 minutes, stirring every 2 minutes.

Form 1: REF K1-6-50-Mod / VER 12/27/16 / page 11 of 15

10.Centrifuge the tubes in a microcentrifuge for 1 minute at 2 thousand g (for example, 5 thousand rpm for a MiniSpin microcentrifuge, Eppendorf Manufacturing Corporation).

11. Without capturing the sorbent, remove the supernatant liquid from each tube with a separate tip without a filter of 200 µl, using a vacuum aspirator.

12.Add 300 μl of washing solution 1 to the test tubes, close the lids tightly, and vortex until the universal sorbent is completely resuspended.

13.Centrifuge the tubes in a microcentrifuge for 1 minute at 2 thousand g.

14. Without capturing the sorbent, remove the supernatant liquid from each tube with a separate 200 µl tip without a filter, using a vacuum aspirator.

15.Add 500 μl of washing solution 2 to the test tubes, close the lids tightly, and vortex until the universal sorbent is completely resuspended

16.Centrifuge the tubes in a microcentrifuge for 1 minute at 7 thousand g (for example, 10 thousand rpm for a MiniSpin microcentrifuge, Eppendorf Manufacturing Corporation).

17. Without capturing the sorbent, remove the supernatant liquid from each tube with a separate 200 µl tip without a filter, using a vacuum aspirator.

18.Repeat the washing procedure following paragraphs. 15-16, remove the supernatant completely.

19. Place the test tubes with open lids in a thermostat with a temperature of 64 °C for 5-10 minutes to dry the universal sorbent.

20.Add 50 µl of elution buffer B into the tubes. Mix by vortex until the sorbent is completely resuspended. Place in a thermostat at 64 °C for 5–10 minutes, stirring periodically (once per minute) using a vortex.

21.Centrifuge the tubes in a microcentrifuge for 1 minute at 10 thousand g. The supernatant contains purified DNA. Samples are ready for PCR.

Purified DNA can be stored at a temperature of 2 to 8 °C for a week, at a temperature of minus 24 to minus 16 °C for 6 months and at a temperature Form 1: REF K1-6-50-Mod / VER 12/27/16 / page 12 of 15 not higher than minus 68 °C throughout the year. To do this, it is necessary, without capturing the sorbent, to transfer the supernatant liquid into a new test tube.

SHELF LIFE, TRANSPORTATION AND STORAGE CONDITIONS.

Best before date. 12 months Expired reagent kits cannot be used. The expiration date of opened reagents corresponds to the expiration date indicated on the labels for unopened reagents, unless otherwise stated in the instructions.

Transportation. The reagent kit should be transported at a temperature of 2 to 8 C for no more than 5 days in thermal containers containing cold elements, all types of covered Vehicle. Upon receipt, disassemble in accordance with the specified storage temperatures.

Storage. Store the reagent kit at a temperature of 2 to 25 C, except for the lysis reagent. Store the lysis reagent in the refrigerator at a temperature of 2 to 8 C.

Refrigeration chambers must provide regulated temperature conditions.