Reagent kit for DNA extraction from sperm cells with removal of foreign cells and DNA "Sperm" 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 sodium lauryl sulfate i auxiliary components dlya optimal lysis;
3. Stabilizator - dlya optimal lysis;
4. Precipitation buffer 1 - dlya protein precipitation;
5. Precipitation buffer 2 - dlya precipitation NA;
6. Wash buffer 1 - dlya promyvki nucleic acids ot cell metabolites and salts;
7. Wash buffer 2 - 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.

Download the short instruction "Sperm"

Download the full instruction "Sperm"

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 450 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. Perenesti tubes na led ili v kholodnyy shtativ i inkubirovat v techenie 1 minuty. V sluchae otsutstviya okhladitelnykh elementov inkubirovat at room temperature v techenie 5-10 minut until completely cooled smesi.
Deproteinizatsiya i osazhdenie NA
1. Add v tubes 75 uL osazhdayushchego bufera 1.
2. Intensivno peremeshat tubes on a vortex mixer, sbrosit kapli s pomoshchyu brief centrifugation.
3. Incubate samples v techenie 3-5 minut vo ldu ili v cooling rack. V sluchae otsutstviya okhladitelnykh elementov inkubirovat at room temperature v techenie 5-10 minut.
4. Centrifuge tubes v techenie 5 minut pri 13 tys. ob/min.
5. Akkuratno, ne zadevaya pellet, perenesti 500 uL supernatanta v novuyu tube obemom 1,5-2 mL.
6. Add k obraztsam 500 uL osazhdayushchego bufera 2 i peremeshat s pomoshchyu pyatikratnogo perevorachivaniya tubes.
Optsionalno:
a) dlya uvelicheniya vykhoda DNA rekomenduetsya inkubirovat samples v freezer pri -20°C v techenie 25 minut;
b) dlya oblegcheniya vizualizatsii obrazuyushchegosya osadka NA vozmozhno ispolzovanie soosaditelya NA (#sat RaissolTM).
7. Centrifuge tubes v techenie 5 minut pri 13 tys. ob/min, posle chego udalit supernatant, ne zadevaya pellet.
Nucleic acid washing
1. Add v tubes 700 uL promyvochnogo bufera 1.
2. Intensivno peremeshat tubes on a vortex mixer.
3. Centrifuge tubes v techenie 5 minut pri 13 tys. ob/min, akkuratno, ne zadevaya pellet, udalit supernatant.
4. Add v tubes 700 uL promyvochnogo bufera 2.
5. Repeat punkty 2,3.
6. Vysushit samples s otkrytymi kryshkami v termostate pri 42°C ili v tsentrifuge-kontsentratore pri 30°C until complete ispareniya promyvochnogo bufera.
Nucleic acid elution
1. Add v tubes 50 uL elyuiruyushchego bufera.
2. Incubate samples v termostate pri 55-60°C v techenie 5 minut, periodicheski peremeshivaya on a vortex mixer.

Buffers nabora "Sperm" 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.
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; 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 25-80oS dlya probirok obemom 1,5-2 mL; skorostnaya mikrotsentrifuga dlya probirok obemom 1,5-2 mL do 13 tys. ob/min; shtativ-okhladitel ili ldogenerator.