Paraffin-embedded Tissue DNA Extraction Kit

1. Komponente kompleta reagensa

Tehnički podaci	50T	100T
Mačka. Ne.	SN0251	SN0252
Stupovi za ekstrakciju DNK (set)	50 (set)	100 (set)
Reagent Buffer IV	20 ml	2 × 20 ml
Pufer reagensa c	30 ml	2 × 30ml
Reagent Buffer FF	60 ml	2 × 60ml
Pufer za pranje 1	15 ml	2 × 15 ml
Elucijski pufer	20 ml	20 ml
Proteinaza K	1ml	2x1ml
RNaseA	1ml	2x1ml
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. Proteinase K and 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

 

Paraffin-embedded tissue DNA purification kit provides a rapid and effective DNA purification solution for samples in paraffin sections. Tissues are collected after paraffin dissolution using a dedicated dewaxing solution, and sample DNA is obtained through subsequent extraction processes.

 

This kit can extract sample DNA from paraffin sections within 30 minuta, and the entire purification process does not require toxic reagents such as phenol-chloroform. 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 FF:This buffer should be stored long-term in an environment between 2°C and 8°C.

B. Reagent Buffers IV and C može se taložiti u uvjetima niske temperature. It is recommended to heat at 65°C for 5 minuta. Nakon što se talog otopi, the solution can be used normally.

C. PranjeBuffer 1 should have a specified amount of anhydrous ethanol added before use, as indicated on the reagent bottle label. Check the label with a tick mark to indicate the addition of anhydrous ethanol.

D. Buffer za eluciju je a 0.1X TE otopina with a minimal amount of EDTA. If EDTA affects subsequent experiments, it is advisable to replace the elution buffer with sterile deionized water.

 

1. Sample Processing:

Select 5-10 paraffin sections (1mm-3mm thickness), use scissors to remove excess wax, and cut the embedded tissue sample into small pieces. Place them in a 1.5ml centrifuge tube, dodati 800μl Reagent Buffer FF, incubate at 75°C for 5 minutes until all paraffin is dissolved. Centrifuge at 12000rpm for 5 minuta, discard the supernatant.

2. Dodati 400μl Reagent Buffer IV, 20μl Proteinase K (10 mg/ml), and 10μl RNaseA (10 mg/ml) to the precipitate from the previous step. Digest at 65°C, stirring every 6-7 times until digestion is complete.
3. Add an equal volume of Pufer reagensa ci an equal volume of anhydrous ethanol, mix thoroughly by pipetting.

(For example: If you add 400μl Reagent Buffer IV, approximately add 430μl Reagent Buffer C and 430μl anhydrous ethanol. Some precipitation may occur after adding Reagent Buffer C, but it does not affect subsequent experiments.)

4. Add the obtained liquid to the DNA extraction purification column (or cartridge) (approximately 650-700μl each time), let it stand at room temperature for 2 minuta, centrifuge at over 8,000rpm for 1 minuta. Odbacite prikupljeni otpad i ponovno umetnite cijev za prikupljanje u stupac za pročišćavanje za sljedeći korak.
5. Repeat step 4, adding the remaining liquid to the DNA extraction purification column (or cartridge), centrifuge at over 8,000rpm for 1 minuta, discard the waste and the collection tube.
6. Postavite stupac za pročišćavanje ekstrakcije DNK (or cartridge) into the collection tube, dodati 300μl PranjeBuffer 1, centrifuge at over 8,000rpm for 1 minuta. Discard the waste and reinsert the purification column into the tube for the next step.

(Bilješka: Confirm that anhydrous ethanol has been added to Pranje Buffer 1.)

7. Dodati 500μl PranjeBuffer 1 na stupac za pročišćavanje DNK (or cartridge), centrifuge at 14,000rpm (20000×g) za 2 minuta. Extend centrifugation time if needed for a dryer membrane.
8. Postavite stupac za pročišćavanje ekstrakcije DNK (or cartridge) in a new centrifuge tube, open the lid, incubate at 65°C for 2 minuta. Extend this step if necessary to evaporate ethanol to prevent residual ethanol from affecting downstream experiments.
9. Suspend the addition of 50-100μl Elution Buffer to the membrane, centrifuge at 12,000rpm for 2 minuta.

(Bilješka: 1. Using 50μl Elution Buffer for DNA elution can increase DNA concentration but decreases total DNA yield. 2. The eluted DNA can be reapplied to the DNA extraction purification column, centrifuged at 12000rpm for 2 minutes again to increase DNA yield.)