Reagent kit for DNA extraction from seeds, flour and seedlings "SeedExt" Raissol

The kit includes all reagents required for DNA extraction:
1. Lysis buffer - na osnove detergenta i auxiliary components dlya optimal lysis;
2. Binding buffer - dlya povysheniya sorbtsii NA na filtre kolonki;
3. Wash buffer 1 - dlya promyvki nucleic acids ot cell metabolites and salts
4. Wash buffer 2 - dlya promyvki nucleic acids ot cell metabolites and salts;
5. Wash buffer 3 - dlya promyvki nucleic acids ot cell metabolites and salts;
6. Elution buffer - dlya elyutsii i khraneniya NA, pH=8,9;
7. Proteinase K - dlya bolee bystrogo i polnogo lizisa;
8. Spin-kolonki - dlya filtratsii reaktsionnoy smesi i sorbtsii NA.

DNA extraction procedure

Probopodgotovka
1. Etap probopreparation obraztsa otlichaetsya v zavisimosti ot vida biomateriala:
Pri rabote s mukoy:
1.a. Vnesti v tube obemom 1,5-2 mL 50 mg naveski obraztsa, add 500 uL liziruyushchego bufera i 20 uL Proteinazy K.
Pri rabote s semenami:
1.b. Izmelchit samples do sostoyaniya muki melkogo pomola. Vnesti v tube obemom 1,5-2 mL 50 mg naveski, add 500 uL liziruyushchego bufera i 20 uL Proteinazy K.
Pri rabote s prorostkami:
1.c. Pomestit navesku 15-50 mg zelenoy chasti prorostka v tube obemom 1,5 mL i intensivno pereteret sample s pomoshchyu spetsialnogo pestika dlya 1,5 mL mikrotsentrifuzhnykh probirok do gomogennogo sostoyaniya. Add 500 uL liziruyushchego bufera. Vnesti 20 uL Proteinazy K.
Primechanie: Pri rabote na avtomaticheskom gomogenizatore trebuemaya naveska obraztsa pomeshchaetsya v spetsialnuyu 1,5-2 mL tube s keramicheskimi ili metallicheskimi businami dlya gomogenizatsii. Intensivnost programmy gomogenizatsii dolzhna byt shchadyashchaya, ne dopuskaya peregrevaniya obraztsov.
2. Intensivno peremeshat tubes on a vortex mixer, sbrosit kapli s pomoshchyu brief centrifugation.
Lysis
1. Incubate samples v termostate pri 56-60℃ v techenie 1-2 chasov, periodicheski vstryakhivaya.
Optsionalno: za 10 minut do okonchaniya lizisa vozmozhno ispolzovanie RNAazy A lyubogo proizvoditelya v kolichestve, rekomendovannom proizvoditelem.
2. Pomestit tubes na led ili v kholodnyy shtativ i inkubirovat v techenie 3 minut. V sluchae otsutstviya okhladitelnykh elementov inkubirovat at room temperature v techenie 10 minut until completely cooled smesi.
Sorbtsiya NA
1. Centrifuge tubes v techenie 5 minut pri 13 tys. ob/min.
2. Perenesti 300 uL supernatanta v novye tubes obemom 1,5 mL.
3. Add 300 uL svyazyvayushchego bufera, mix thoroughly tubes on a vortex mixer, sbrosit kapli s pomoshchyu brief centrifugation.
4. Perenesti ves obem smesi v spin-kolonku.
5. Centrifuge tubes v techenie 1 minuty pri 13 tys. ob/min. Udalit filtrat iz sobiratelnykh probirok.
Nucleic acid washing
1. Add v tubes 650 uL promyvochnogo bufera 1.
2. Centrifuge tubes v techenie 1 minuty pri 13 tys. ob/min. Udalit filtrat iz sobiratelnykh probirok.
3. Add v tubes 650 uL promyvochnogo bufera 2.
4. Centrifuge tubes v techenie 30 sekund pri 13 tys. ob/min. Udalit filtrat iz sobiratelnykh probirok.
5. Add v tubes 650 uL promyvochnogo bufera 3.
6. Centrifuge tubes v techenie 30 sekund pri 13 tys. ob/min. Udalit filtrat iz sobiratelnykh probirok.
7. Repeat p 5-6.
8. Vernut kolonki v sobiratelnye tubes. Centrifuge tubes v techenie 1 minuty pri maks. ob/min.
Nucleic acid elution
1. Perenesti spin-kolonki v novye tubes obemom 1,5-2 mL. Sobiratelnye tubes utilizirovat.
2. Nanesti na membranu spin-kolonok 50-100 uL elyuiruyushchego bufera. Incubate v techenie 5 minut.
3. Centrifuge tubes v techenie 30 sekund pri maks. ob/min.
4. Utilizirovat spin-kolonki.
Optsionalno: dlya povysheniya vykhoda NA filtrat neobkhodimo povtorno nanesti na membranu kolonki i/ili ispolzovat nagretyy do 60-70℃ elyuiruyushchiy bufer.
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 corresponds to ≥1,7.

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Buffers nabora "SeedExt" can be stored pri temperature ot +4 do +25℃ for 12 months from the manufacturer's release date. If a precipitate is present pri minimalnykh temperaturakh khraneniya, warm the solutions to room temperature.
Enzyme Proteinase K must be stored pri -20℃, shelf life is 12 mesyatsev. A short-term storage temperature increase is allowed (transportation) fermenta Proteinase K ot +4℃ do +25℃ for no more than 5 days.

1. Microcentrifuge tubes obemom 1,5-2 mL;
2. Rack for microcentrifuge tubes obemom 1,5-2 mL;
3. Variable-volume pipettes na 20-200 uL, 100-1000 uL i sootvetstvuyushchie disposable tips;
4. Spin-kolonki s sobiratelnymi probirkami (idut v komplekte);
5. Vorteks;
6. Dry block thermostat s vozmozhnostyu podderzhaniya temperaturnogo rezhima v diapazone 25-80ºS dlya probirok obemom 1,5-2 mL;
7. High-speed microcentrifuge dlya probirok obemom 1,5-2 mL do 13 tys. ob/min;
8. Analiticheskie vesy s tochnostyu vzveshivaniya 0,1 mg;
9. Pestik dlya gomogenizatsii v 1,5 mL probirkakh.

Primechanie: pri gomogenizatsii obraztsov v farforovoy stupke s pestikom neobkhodimo ispolzovat nabor "SeedExt+".