Stool or Soil Genomic DNA Extraction Kit

1. Komponente kompleta reagensa

Tehnički podaci	50T	100T
Mačka. Ne.	SN0241	SN0242
Stupovi za ekstrakciju DNK (set)	50 (set)	100 (set)
Humic acid removal reagent I	50ml	2x50ml
Humic acid removal reagent II	50ml	2x50ml
Reagent Buffer Ⅳ	30 ml	2×30 ml
Reagent Buffer C plus	30 ml	2×30 ml
Inhibitor removal buffer	30ml	2x30ml
Lizocim	1ml	2x1ml
Proteinaza K	1ml	2x1ml
RNaza A	1ml	2x1ml
Pufer za pranje 1	15 ml	2 × 15 ml
Elucijski pufer	20 ml	2 ×20 ml
Priručnik za upute	1	1

2. Skladištenje

Ovaj se komplet reagensa treba čuvati na sobnoj temperaturi (15-25℃) I u suhim uvjetima, s rokom trajanja 12 mjeseca. Stupovi za pročišćavanje DNK mogu se pohraniti za 1 godina u hladnom i suhom okruženju. Lizocim, Proteinaza K, i RNaza A contain preservatives, omogućavajući prijevoz na sobnoj temperaturi, Ali za dugoročno skladištenje, Treba ih držati na -20 ℃.

3. Upute za korištenje kompleta reagensa

3.1 Ovaj komplet reagensa namijenjen je istraživanju molekularne biologije i ne smije se koristiti za dijagnozu ili liječenje bolesti.

3.2 Neke komponente u kompletu reagensa sadrže iritante. Preporučuju se zaštitne mjere poput nošenja zaštitne odjeće i naočala.

3.3 Tijekom upotrebe ovog kompleta reagensa, brza centrifuga, vodena kupelji (metalna kupka), vrtlog miksera, bezvodni etanol, sterilna deionizirana voda, i EP epruvete treba pripremiti korisnik.

4. Uvod u komplet reagensa

This kit provides a fast and efficient method for purifying microbial genomic DNA from soil, feces, and vomit samples. It is widely used for microbial DNA extraction from soil, feces, and vomit.

 

This kit effectively removes sample impurities and inhibitors, reducing inhibitory reactions in downstream experiments such as PCR. Cijeli postupak pročišćavanja ne zahtijeva toksične reagense kao što je fenol-kloroform. The extracted DNA can be directly used for PCR, Južni blot, and other applications.

5. Eksperimentalni principi i postupci

6. Ekstrakcijski postupak

Prije početka eksperimenta:

A. Reagent Buffer IV može se taložiti u uvjetima niske temperature. Preporučujemo grijanje na 65 ℃ za 5 minuta. Nakon što se talog otopi, Može se normalno koristiti.

B. Prije upotrebe, Dodajte navedenu količinu bezvodnog etanola u Pufer za pranje 1 Kao što je naznačeno na etiketi boca. Označite ček na etiketi kako biste naznačili dodavanje bezvodnog etanola.

C. Buffer za eluciju je a 0.1X TE otopina Sadrži minimalne količine EDTA. Ako EDTA može utjecati na naknadne eksperimente, Preporučljivo je zamijeniti pufer za eluciju sterilnom deioniziranom vodom.

1. Sample Processing (Inhibitor Removal):

A. Fecal and Vomit Samples: Add approximately 200mg of the sample to be extracted, dodati 1ml of Humic Acid Removal Reagent I, vortex thoroughly for 1-2 minuta, centrifuga na 12,000 RPM za 2 minuta, discard the supernatant, dodati 560μl of Buffer IV to the pellet, invert and mix thoroughly, digest at 85℃ for 5 minuta, invert and mix 6-7 times during digestion, then proceed to step 2.

B. Soil Samples: Weigh approximately 0.1g-0.5g of sieved soil, dodati 500μl of Buffer IV and 100μl of Humic Acid Removal Reagent I, invert and mix thoroughly, digest at 85℃ for 5 minuta, invert and mix 6-7 times during digestion, then proceed to step 2.

(Bilješka: Humic Acid Removal Reagent I/Humic Acid Removal Reagent II is mainly used for soils with high humic acid content, such as forest soils and sedimentary soils. Dodati 1ml of Humic Acid Removal Reagent I to the sample, vortex, shake for 1-2 minuta, centrifuga na 8,000 RPM za 2 minuta, discard the supernatant, then add 1ml of Humic Acid Removal Reagent II, vortex, centrifuga na 8,000 RPM za 2 minuta, discard the supernatant. This step can further reduce soil humic acid inhibitors but significantly reduce DNA yield.)

2. Centrifuga na 12,000 RPM za 2 minuta, transfer the supernatant to a new centrifuge tube (approximately 500μl liquid transferred), add 20μl Proteinase K (10 mg/ml), 20μl Lysozyme, and 20μl RNase A, and incubate at 65℃ for 2 minuta.
3. Add an equal volume of Reagens Buffer C plus (approximately 560μl liquid transferred) to the lysate, Dobro promiješajte. Ako se pojavi bijeli talog, let it settle; it won’t affect subsequent experiments after the precipitate disappears.
4. Add an equal volume of ethanol to Reagens Buffer C plus (Npr., dodati 560μl of Reagens Buffer C plus, then add 560μl of ethanol), Dobro promiješajte. There may be precipitation, but it won’t affect subsequent experiments.
5. Add the obtained liquid to the DNA extraction purification column (sleeve) (approximately 650-700μl each time), centrifuge at over 8,000 RPM za 1 minuta, discard the collected waste liquid, and reinsert the collection tube into the DNA extraction purification column (sleeve) for the next step.
6. Dodati 400μl Inhibitor Removal Buffer, centrifuge at over 8,000 RPM za 1 minuta, discard the waste liquid, and reinsert the DNA purification column into the sleeve for the next step.
7. Dodati 500μl Wash Buffer 1 na stupac za pročišćavanje DNK (sleeve), centrifuga na 14,000 rpm (20,000×g) za 1 minuta.
8. Dodati 300μl Wash Buffer 1 again to the DNA extraction purification column (sleeve), centrifuga na 14,000 rpm (20,000×g) za 2 minuta.
9. Postavite stupac za pročišćavanje ekstrakcije DNK (sleeve) u novu cijev za centrifugu, open the lid, and incubate at 65℃ for 2 minuta. This step can be extended if necessary to evaporate ethanol as much as possible to prevent ethanol residue from affecting downstream experiments.
10. Drip 100μl Elution Buffer onto the membrane, centrifuga na 12,000 RPM za 2 minuta.

(Bilješka: 1. Eluting DNA with 50μl Elution Buffer can increase DNA concentration but reduce total DNA yield; 2. The eluted DNA can be reapplied to the DNA extraction purification column, centrifuged again at 12,000 RPM za 2 minuta, and collected to increase DNA yield.)