Whole-genome library preparation kit for fragments over 300 bp "SG GM Maxi" Raissol

V nabor vklyucheny vse neobkhodimye reagenty dlya prigotovleniya polnogenomnykh libraries:
1. 3v1 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.

Download the short instruction "Shot Gun GM Maxi" (pdf)

Download the full instruction "Shot Gun GM Maxi" (pdf)

Provedenie protsedury prigotovleniya polnogenomnykh libraries

Fragmentatsiya
1. Vnesti 10 uL obraztsa DNA s kontsentratsiey 30 ng/uL v tubes v stripakh libo v 96-lunochnyy planshet obemom 0,2 mL. Kontsentratsiya dolzhna byt ustanovlena: spektrofotometricheski pri dline volny 260 nm.
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: rekomendovannaya programma rasschitana na sredniy razmer produkta 300-350 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 35 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 35 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.

Vse reagenty nabora "SG GM Maxi" must be stored pri temperature ot -20℃ do -15℃, shelf life is 12 mesyatsev.
Poluchennye polnogenomnye librariesi can be stored pri +4℃ v techenie sutok ili pri -20℃ bolee dlitelnyy srok.

Required materials and equipment:

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;
7. Amplifikator; magnitnyy shtativ planshetnogo tipa dlya probirok obemom 0,2 mL.

Neobkhodimye reagenty:

1. Magnitnye chastitsy dlya selektivnoy cleanup DNA, naprimer, "Smart beads" Raissol™ (#smartb 50/120/240), ili analogi tekhnologii SPRI;
2. Deionizirovannaya voda;
3. TE bufer s nizkim soderzhaniem EDTA;
4. 80% etanol;
5. Praymery - komplekt indeksov 1 (#Plate1_SG_GM); praymery - komplekt indeksov 2 (#Plate2_SG_GM).