Reagent kit for metagenomic DNA extraction from fecal samples "Meta Fec" Raissol

The kit includes all reagents required for DNA extraction:

1. Lysis buffer - na osnove chaotropic agents i auxiliary components dlya optimal lysis;
2. Binding buffer 1 - dlya povysheniya sorbtsii NA na magnitnykh chastitsakh;
3. Binding buffer 2 - dlya povysheniya sorbtsii NA na magnitnykh chastitsakh;
4. Wash buffer 1 - dlya promyvki nucleic acids ot organicheskikh primesey i soley;
5. Wash buffer 2 - dlya promyvki nucleic acids ot organicheskikh primesey i soley;
6. Wash buffer 3 - dlya promyvki nucleic acids ot organicheskikh primesey i soley;
7. Wash buffer 4 - dlya promyvki nucleic acids ot organicheskikh primesey i soley;
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.

Lysis

1. V tubes obemom 1,5-2 mL place 100 mg issleduemykh obraztsov fecal samples.
2. Add k obraztsam 400 uL liziruyushchego bufera i 20 uL Proteinazy K.
3. Intensivno peremeshat tubes on a vortex mixer.
4. Incubate tubes v termostate pri temperature 56℃ v techenie 1-2 chasov, periodicheski peremeshivaya.
5. Sbrosit kapli s pomoshchyu brief centrifugation.
6. Pomestit tubes na led ili v kholodnyy shtativ i inkubirovat v techenie 3 minut. V sluchae otsutstviya okhladitelnykh elementov inkubirovat at room temperature v techenie 10 min until completely cooled smesi.
Sorbtsiya NA
1. Add k smesi 350 uL svyazyvayushchego bufera 1.
2. Intensivno peremeshat tubes on a vortex mixer, sbrosit kapli s pomoshchyu brief centrifugation.
3. Incubate at room temperature v techenie 5 minut.
4. Centrifuge tubes v techenie 5 minut pri maks. ob/min.
5. Akkuratno, ne zadevaya pellet, perenesti supernatant v novye tubes.
6. Repeat punkty 4-5.
7. Add v tubes 240 uL svyazyvayushchego bufera 2 i 50 uL magnitnykh chastits, mix thoroughly tubes on a vortex mixer.
8. Incubate at room temperature v techenie 5 minut, periodicheski peremeshivaya.
9. Sbrosit kapli s pomoshchyu brief centrifugation.
10. Incubate na magnitnom shtative v techenie 5 minut.
11. Akkuratno, ne zadevaya pellet chastits, udalit supernatant.

Nucleic acid washing

1. Add v tubes 500 uL promyvochnogo bufera 1.
2. Intensivno peremeshat tubes on a vortex mixer, sbrosit kapli s pomoshchyu brief centrifugation.
3. Incubate tubes na magnitnom shtative v techenie 2 minut. Akkuratno, ne zadevaya pellet chastits, udalit supernatant.
4. Add v tubes 500 uL promyvochnogo bufera 2.
5. Repeat punkty 2-3.
6. Add v tubes 500 uL promyvochnogo bufera 3.
7. Intensivno peremeshat tubes on a vortex mixer, sbrosit kapli s pomoshchyu brief centrifugation.
8. Incubate tubes na magnitnom shtative v techenie 2 minut. Akkuratno, ne zadevaya pellet chastits, udalit supernatant.
9. Repeat punkty 6-8.
10. Add v tubes 500 uL promyvochnogo bufera 4.
11. Repeat punkty 7-8.
12. Vysushit samples s otkrytymi kryshkami v termostate pri temperature 56-60℃ v techenie 5-10 minut until complete ispareniya promyvochnogo rastvora.

Nucleic acid elution

1. Add v tubes 50-100 uL elyuiruyushchego bufera.
2. Intensivno peremeshat on a vortex mixer, sbrosit kapli s pomoshchyu brief centrifugation.
3. Incubate v termostate pri temperature 56-60℃ v techenie 10 minut, periodicheski peremeshivaya.
4. Sbrosit kapli s pomoshchyu brief centrifugation.
5. Incubate tubes na magnitnom shtative v techenie 3-5 minut.
6. Akkuratno, ne zadevaya chastitsy, perenesti elyuat v novye tubes.
Optsionalno: rekomenduetsya provesti dopolnitelnoe tsentrifugirovanie elyuata na maksimalnoy skorosti dlya udaleniya ostatochnogo kolichestva magnitnykh chastits.

Poluchennye rastvory nucleic acids can be stored do 7 dney pri temperature ot +2℃ do +4℃ i do dvukh let pri temperature -20℃. Po absorbance ratio na A260/A280 purity of the resulting solution DNA corresponds to ≥1,7.

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Buffers nabora "Meta Fec" can be stored pri temperature ot +4℃ do +25℃ for 12 months from the manufacturer's release date. If a precipitate is present pri minimalnykh temperaturakh khraneniya, warm the solutions to room temperature.
Magnitnye chastitsy can be stored pri temperature ot +4℃ do +25℃ for 12 months from the manufacturer's release date. Pered applicationm suspenziyu chastits neobkhodimo tshchatelno vzbaltyvat do odnorodnogo sostoyaniya.
Enzyme Proteinase K must be stored pri -20℃, shelf life is 12 mesyatsev. A short-term storage temperature increase is allowed (transportation) fermenta Proteinase K ot +4℃ do +25℃ for no more than 5 days.

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