DNA extraction procedure
Lysis1. V tubes obemom 1,5-2 mL place 300 uL issleduemykh prob milk.
2. Add k obraztsam 100 uL liziruyushchego bufera i 20 uL Proteinazy K.
3. Intensivno peremeshat tubes on a vortex mixer.
4. Incubate v termostate pri temperature 56-60℃ v techenie 30-60 minut, periodicheski peremeshivaya.
5. Sbrosit kapli s pomoshchyu brief centrifugation.
Sorbtsiya NA1. Add v tubes 800 uL svyazyvayushchego bufera i 50 uL magnitnykh chastits.
2. Incubate tubes v techenie 10-15 minut at room temperature, periodicheski peremeshivaya.
3. Sbrosit kapli s pomoshchyu brief centrifugation.
4. Incubate tubes na magnitnom shtative v techenie 2 minut.
5. Akkuratno, ne zadevaya chastitsy, udalit supernatant.
Nucleic acid washing1. 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 3 minut, posle chego akkuratno, ne zadevaya chastitsy, udalit supernatant.
4. Add v tubes 500 uL promyvochnogo bufera 2.
5. Intensivno peremeshat tubes on a vortex mixer, sbrosit kapli s pomoshchyu brief centrifugation.
6. Incubate tubes na magnitnom shtative v techenie 3 minut, posle chego akkuratno, ne zadevaya chastitsy, udalit supernatant.
7. Repeat punkty 4-6.
8. Add v tubes 500 uL promyvochnogo bufera 3.
9. Repeat punkty 5-6.
10. Vysushit samples s otkrytymi kryshkami v termostate pri 56-60℃ v techenie 5-10 minut until complete ispareniya promyvochnogo rastvora.
Nucleic acid elution1. Add v tubes 40-60 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 5 minut, periodicheski vstryakhivaya.
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 sootvetstvuyut ≥1,7.
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