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Amplicon library preparation kit "SG GM Ampli" Raissol

Raissol
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Amplicon library preparation kit "SG GM Ampli" Raissol is a Raissol reagent product for ngs library preparation in molecular biology, genetics, diagnostic and research laboratories.

Application area

Used for sample preparation, nucleic acid analysis, PCR/qPCR workflows and sequencing-related projects where applicable.

Sample types

The product is intended for work with biological material.

Advantages of Raissol reagents

  • stable reagent performance and reproducible laboratory results
  • compatibility with common molecular biology protocols
  • convenient workflow for routine and research procedures
  • efficient preparation of biological material for downstream analysis

Key specifications

CategorySequencing library preparation kits;Agrogenetics>>>Agrogenetics - library preparation;Raissol
ManufacturerRaissol
Primary useNGS library preparation
Sample typebiological material

Section: Kity dlya prigotovleniya DNA libraries

Kit contents
Instructions for use
Storage conditions
Required materials and equipment

Kit contents

V nabor vklyucheny vse neobkhodimye reagenty dlya prigotovleniya libraries iz amplikonov:
1. 3v1 bufer;
2. 3-ferment A;
3. Ligaznyy bufer;
4. Ligaza;
5. Adaptery;
6. PCR bufer;
7. PCR ferment 1;
8. PCR ferment 2.

Instructions for use


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

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


Provedenie protsedury prigotovleniya libraries

Reparatsiya

1. Vnesti 10 uL ochishchennykh fragmentov DNA s kontsentratsiey ot 1 do 10 ng/uL v tubes v stripakh, libo v 96-lunochnyy planshet obemom 0,2 mL. Kontsentratsiya dlya amplikonov dolzhna byt ustanovlena spektrofotometricheski s applicationm interkalyatorov (#spectrahs-100/500/1000; #spectrabr-100/500/1000).

2. Prigotovit master-miks na neobkhodimoe kolichestvo reactions:

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

4. Vnesti 15 uL master-miksa k obraztsam.

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

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

Ligirovanie

1. Prigotovit master-miks na neobkhodimoe kolichestvo reactions:

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

3. Posle zaversheniya programmy reparatsii, 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. K obraztsam, soderzhashchim fragmenty <150 bp, add 60 uL magnitnykh chastits, k obraztsam, soderzhashchim fragmenty >150 bp, add 50 uL magnitnykh chastits, mix thoroughly on a vortex mixer do gomogennogo sostoyaniya, sbrosit kapli s pomoshchyu brief centrifugation i inkubirovat v techenie 5 minut at room temperature.

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

3. Akkuratno, ne zadevaya chastitsy, 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°S until complete ispareniya spirta.

6. Add 24 uL deionizirovannoy vody k magnitnym chastitsam. 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:

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. K obraztsam, soderzhashchim fragmenty <150 bp, add 60 uL magnitnykh chastits, k obraztsam, soderzhashchim fragmenty >150 bp, add 50 uL magnitnykh chastits, mix thoroughly on a vortex mixer do gomogennogo sostoyaniya, sbrosit kapli s pomoshchyu brief centrifugation i inkubirovat v techenie 5 minut at room temperature.

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

3. Akkuratno, ne zadevaya chastitsy, udalit supernatant.

4. Add 180 uL 80% etanola k kazhdomu obraztsu, inkubirovat v techenie 30 sekund, peremeshchaya tubes na magnitnom shtative. 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°S 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 DNA librariesi can be stored pri +4℃ v techenie sutok ili pri -20℃ bolee dlitelnyy srok. DNA librariesi mogut byt ispolzovany v dalneyshey podgotovke k sekvenirovaniyu.

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Storage conditions

Vse komponenty nabora "SG GM Ampli" must be stored pri -20°S, shelf life is 12 mesyatsev.

Required materials and equipment

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 libo dlya 96-lunochnykh planshetov;
  7. Amplifikator;
  8. Magnitnyy shtativ.
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);
  6. Praymery - komplekt indeksov 2 (#Plate2_SG_GM).