ShotGun Plus whole-genome library preparation kit with magnetic beads "SG GM Plus" Raissol

Provedenie protsedury prigotovleniya polnogenomnykh libraries

Fragmentatsiya

1. Vnesti 10 uL obraztsa DNA s kontsentratsiey 20 ng/uL (dlya genomnoy DNA) ili 10 ng/uL (dlya amplikonov) v tubes v stripakh libo v 96-lunochnyy planshet obemami 0,2 mL. Kontsentratsiya dolzhna byt ustanovlena: 1) dlya genomnoy DNA - spektrofotometricheski pri dline volny 260 nm 2) dlya amplikonov - spektrofotometricheski s applicationm interkalyatorov (#spectrahs-100/500/1000; #spectrabr-100/500/1000).

2. Prigotovit master-miks na neobkhodimoe kolichestvo reactions:

3v1 bufer - 13,4 uL na 1 reaktsiyu.

3-ferment - 1,6 uL na 1 reaktsiyu.

DNA-nukleaza - 1 uL na 1 reaktsiyu.

3. Mix master-miks, sbrosit kapli s pomoshchyu brief centrifugation.

4. Vnesti 16 uL master-miksa k obraztsam.

5. Pomestit tubes v amplifikator s predvaritelno ustanovlennoy programmoy dlya fragmentatsii posle vykhoda pribora v rabochee sostoyanie 1-go etapa:

6. Posle ustanovki probirok v amplifikator neobkhodimo propustit pervyy etap.

Primechanie: pri ispolzovanii DNA-nukleazy dlya fragmentirovaniya DNA na neobkhodimyy razmer, sleduet provesti predvaritelnoe testirovanie vremeni fragmentatsii (polka 2: +65℃) s shagom 2,5 minuty dlya ustanovleniya optimalnogo razmera (umenshenie vremeni privodit k uvelicheniyu razmera produkta). Rekomendovannaya programma rasschitana na sredniy razmer produkta 140-160 bp

Ligirovanie

1. Prigotovit master-miks na neobkhodimoe kolichestvo reactions:

Ligaznyy bufer - 20,5 uL na 1 reaktsiyu.

Adaptery - 3,5 uL na 1 reaktsiyu.

Ligaza - 1 uL na 1 reaktsiyu.

2. Mix master-miks, sbrosit kapli s pomoshchyu brief centrifugation.

3. Posle zaversheniya programmy fragmentatsii vnesti 25 uL master-miksa k obraztsam.

4. Ustanovit programmu dlya ligirovaniya. Pomestit tubes v amplifikator s predvaritelno ustanovlennoy programmoy dlya ligirovaniya posle vykhoda pribora v rabochee sostoyanie 1-go etapa:

5. Posle ustanovki probirok v amplifikator neobkhodimo propustit pervyy etap.

Ochistka posle ligirovaniya

1. Add 45 uL magnitnykh chastits k kazhdomu obraztsu, mix thoroughly on a vortex mixer do gomogennogo sostoyaniya, inkubirovat v techenie 5 minut at room temperature, sbrosit kapli s pomoshchyu brief centrifugation.

2. Pomestit samples na magnitnyy shtativ, inkubirovat v techenie 3-5 minut do prozrachnosti rastvora.

3. Akkuratno, ne zadevaya pellet chastits, udalit supernatant.

4. Povtorno tsentrifugirovat samples na maksimalnykh oborotakh tsentrifugi v techenie 10-20 s.

5. Pomestit samples na magnitnyy shtativ, akkuratno, ne zadevaya pellet chastits, maksimalno udalit ostatki supernatanta.

6. Add 24 uL elyuiruyushchego bufera k osadku chastits. Intensivno peremeshat tubes on a vortex mixer, sbrosit kapli s pomoshchyu brief centrifugation.

7. Pomestit samples na magnitnyy shtativ, inkubirovat v techenie 2-3 minut do prozrachnosti rastvora.

8. Perenesti 22 uL elyuata v novye tubes, ne zakhvatyvaya magnitnye chastitsy.

Postanovka indeksnoy PCR

1. Add k kazhdomu obraztsu po 2 uL individualnogo praymernogo miksa iz plansheta s praymerami (#Plate1_SG_GM/#Plate2_SG_GM).

2. Prigotovit master-miks na neobkhodimoe kolichestvo reactions:

PCR bufer - 25 uL na 1 reaktsiyu.

PCR ferment 1 - 0,5 uL na 1 reaktsiyu.

PCR ferment 2 - 0,75 uL na 1 reaktsiyu.

3. Mix master-miks, sbrosit kapli s pomoshchyu brief centrifugation.

4. Vnesti po 26,25 uL master-miksa k obraztsam.

5. Pomestit tubes v amplifikator. Zapustit programmu dlya indeksnoy PCR:

Ochistka posle indeksnoy PCR

1. Add 50 uL magnitnykh chastits k kazhdomu obraztsu, mix thoroughly on a vortex mixer do gomogennogo sostoyaniya, inkubirovat v techenie 5 minut at room temperature, sbrosit kapli s pomoshchyu brief centrifugation.

2. Pomestit samples na magnitnyy shtativ, inkubirovat v techenie 3-5 minut do prozrachnosti rastvora.

3. Akkuratno, ne zadevaya pellet chastits, udalit supernatant.

4. Add 180 uL 80% etanola k kazhdomu obraztsu, inkubirovat v techenie 30 sekund, peremeshchaya tubes na magnitnom shtative. Akkuratno, ne zadevaya pellet chastits, udalit supernatant.

5. Repeat punkt 4. Vysushit samples s otkrytymi kryshkami at room temperature v techenie 10 minut ili pri +37°S until complete ispareniya spirta.

6. Add 22 uL elyuiruyushchego bufera k osadku chastits. Intensivno peremeshat tubes on a vortex mixer, sbrosit kapli s pomoshchyu brief centrifugation.

7. Pomestit samples na magnitnyy shtativ, inkubirovat v techenie 2-3 minut do prozrachnosti rastvora.

8. Perenesti 20 uL elyuata v novye tubes.

Poluchennye polnogenomnye librariesi can be stored pri +4℃ v techenie sutok ili pri -20℃ bolee dlitelnyy srok. Polnogenomnye librariesi mogut byt ispolzovany v dalneyshey podgotovke k sekvenirovaniyu.

Download the short instruction "SG GM Plus" (pdf)

Download the full instruction "SG GM Plus" (pdf)

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1. Microcentrifuge tubes obemom 1,5-2 mL;
2. Rack for microcentrifuge tubes obemom 1,5-2 mL;
3. Probirki v stripakh libo 96-lunochnyy planshet obemami 0,2 mL;
4. Variable-volume pipettes na 0,5-10 uL, 2-20 uL, 20-200 uL, 100-1000 uL i sootvetstvuyushchie disposable tips;
5. Vorteks;
6. Tsentrifuga dlya probirok v stripakh obemom 0,2 mL libo dlya 96-lunochnykh planshetov;
7. Amplifikator;
8. Magnitnyy shtativ planshetnogo tipa dlya probirok obemom 0,2 mL.

1. 80% etanol;
2. Praymery - komplekt indeksov 1 (#Plate1_SG_GM),
3. Praymery - komplekt indeksov 2 (#Plate2_SG_GM).