Reagent kit for DNA extraction from sperm cells with removal of foreign cells and DNA "Sperm M" Raissol

The kit includes all reagents required for DNA extraction:
1. Lysis buffer 1 - dlya udaleniya primesi chuzherodnykh kletok i DNA;
2. Lysis buffer 2 - na osnove detergentov i auxiliary components dlya optimal lysis;
3. Stabilizator - dlya optimal lysis;
4. Binding buffer - dlya sorbtsii NA na magnitnykh chastitsakh;
5. Wash buffer 1 - dlya promyvki nucleic acids ot cell metabolites and salts;
6. Wash buffer 2 - dlya promyvki nucleic acids ot cell metabolites and salts;
7. Wash buffer 3 - dlya promyvki nucleic acids ot cell metabolites and salts;
8. Elution buffer - dlya elyutsii i khraneniya NA, pH=8,9;
9. Proteinase K - dlya bolee bystrogo i polnogo lizisa;
10. Magnitnye chastitsy - dlya sorbtsii NA.

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Probopodgotovka
Etap probopreparation obraztsa otlichaetsya v zavisimosti ot vida biomateriala:
a) Pri rabote s osadkom sperm cells:
Probopodgotovka ne trebuetsya, pereyti k etapu lizisa.
b) Pri rabote s obraztsami semennoy zhidkosti:
Vnesti v tubes obemom 1,5-2 mL 30-300 uL obraztsa. Centrifuge tubes v techenie 5 minut pri 13 tys. ob/min, akkuratno, ne zadevaya pellet, polnostyu udalit supernatant.
c) Pri rabote s obraztsami semennoy zhidkosti, kontaminirovannymi chuzherodnymi kletkami (epiteliy vlagalishcha, bukkalnyy epiteliy i dr.):
1) V tubes obemom 1,5-2 mL place issleduemye samples, add 200 uL liziruyushchego bufera 1 i 20 uL Proteinazy K. Intensivno peremeshat samples on a vortex mixer, sbrosit kapli s pomoshchyu brief centrifugation. Incubate samples pri 60°C v techenie 15 minut, peremeshivaya kazhdye 3-5 minut. Zatem tsentrifugirovat samples v techenie 5 minut pri 13 tys. ob/min, akkuratno, ne zadevaya pellet, polnostyu udalit supernatant.
2) Repeat punkt 1.
3) Add k poluchennym osadkam 400 uL liziruyushchego bufera 1. Intensivno peremeshat samples on a vortex mixer. Centrifuge tubes v techenie 5 minut pri 13 tys. ob/min, akkuratno, ne zadevaya pellet, polnostyu udalit supernatant.
Lysis
1. K osadkam sperm cells add 400 uL liziruyushchego bufera 2, 20 uL Proteinazy K i 2 uL stabilizatora. Vorteksirovat samples until complete razrusheniya konglomerata kletok, sbrosit kapli s pomoshchyu brief centrifugation.
2. Incubate samples v termostate pri 60°C v techenie 1-2 chasov, periodicheski peremeshivaya.
3. Add k obraztsam 300 uL svyazyvayushchego bufera. Intensivno peremeshat tubes on a vortex mixer.
4. Centrifuge samples v techenie 3 minut pri maks. ob/min. Akkuratno, ne zadevaya pellet, perenesti maksimalnyy obem supernatanta v novye tubes obemom 1,5-2 mL.
Sorbtsiya NA
1. Add v tubes 300 uL svyazyvayushchego bufera i 100 uL magnitnykh chastits.
2. Intensivno peremeshat tubes on a vortex mixer, sbrosit kapli s pomoshchyu brief centrifugation.
3. Incubate samples at room temperature v techenie 5 minut, periodicheski peremeshivaya.
4. Sbrosit kapli s pomoshchyu brief centrifugation.
5. Incubate samples na magnitnom shtative v techenie 3 minut. Akkuratno, ne zadevaya magnitnye chastitsy, udalit supernatant.
Nucleic acid washing
1. Add 600 uL promyvochnogo bufera 1.
2. Intensivno peremeshat tubes on a vortex mixer, sbrosit kapli s pomoshchyu brief centrifugation.
3. Incubate samples na magnitnom shtative v techenie 2 minut, posle chego akkuratno, ne zadevaya magnitnye chastitsy, udalit supernatant.
4. Add 600 uL promyvochnogo bufera 2.
5. Repeat punkty 2-3.
6. Add 600 uL promyvochnogo bufera 3.
7. Repeat punkty 2-3.
8. Vysushit samples s otkrytymi kryshkami v termostate pri 56-60°C ili at room temperature until complete ispareniya promyvochnogo bufera 3.
Nucleic acid elution
1. Add v tubes 50 uL elyuiruyushchego bufera.
2. Intensivno peremeshat tubes on a vortex mixer, sbrosit kapli s pomoshchyu brief centrifugation.
3. Incubate samples v termostate pri 60°C v techenie 10 minut, periodicheski peremeshivaya.
4. Cbrosit kapli s pomoshchyu brief centrifugation.
5. Incubate samples na magnitnom shtative v techenie 3 minut.
6. Perenesti elyuat v novye tubes obemom 1,5-2 mL.
Optsionalno: rekomenduetsya provesti dopolnitelnoe tsentrifugirovanie elyuata na maksimalnoy skorosti dlya udaleniya ostatochnogo kolichestva magnitnykh chastits.

Buffers nabora "Sperm M" can be stored pri temperature ot +4°C do +25°C for 12 months c daty vypuska izgotovitelya. If a precipitate is present pri minimalnykh temperaturakh khraneniya, warm the solutions to room temperature.
Magnitnye chastitsy can be stored pri temperature ot +4°C do +25°C for 12 months c daty vypuska izgotovitelya. Suspenziyu chastits neobkhodimo tshchatelno vzbaltyvat do odnorodnogo sostoyaniya pered applicationm.
Stabilizator must be stored pri temperature ot +2°C do +4°C for 12 months from the manufacturer's release date.
Enzyme Proteinase K khranitsya pri -20°C, shelf life is 12 mesyatsev. A short-term storage temperature increase is allowed (transportation) ot +4°C do +25°C for no more than 5 days.

Microcentrifuge tubes obemom 1,5-2 mL; magnitnyy shtativ dlya mikrotsentrifuzhnykh probirok obemom 1,5-2 mL; avtomaticheskie dozatory peremennogo obema na 2-20 uL, 20-200 uL, 100-1000 uL i sootvetstvuyushchie disposable tips; vortex mixer; tverdotelnyy termostat s vozmozhnostyu podderzhaniya temperaturnogo rezhima v diapazone 25-80 S dlya probirok obemom 1,5-2 mL; skorostnaya mikrotsentrifuga dlya probirok obemom 1,5-2 mL do 13 tys. ob/min.