ShotGun whole-genome library preparation kit "SG GM" Raissol

V nabor vklyucheny vse neobkhodimye reagenty dlya prigotovleniya polnogenomnykh libraries:
1. 3 v 1 bufer;
2. 3-ferment;
3. DNA-nukleaza;
4. Ligaznyy bufer;
5. Ligaza;
6. Adaptery;
7. PCR bufer;
8. PCR ferment 1;
9. PCR ferment 2.

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 obemom 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. Add 180 uL 80% etanola k kazhdomu obraztsu, inkubirovat v techenie 30 sekund, peremeshchaya tubes na magnitnom shtative, menyaya ikh polozhenie otnositelno magnita. Akkuratno, ne zadevaya chastitsy, udalit supernatant.
5. Repeat punkt 4. Vysushit samples s otkrytymi kryshkami at room temperature v techenie 10 minut ili pri +37℃ until complete ispareniya spirta.
6. Add 24 uL deionizirovannoy vody 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℃ until complete ispareniya spirta.
6. Add 22 uL TE buffera s nizkim soderzhaniem EDTA 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.

Skachat kratkuyu instruktsiyu "ShotGun GM" (pdf)

Download the full instruction "ShotGun GM" (pdf)

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Vse reagenty nabora "SG GM" must be stored pri -20℃, shelf life is 12 mesyatsev.