Navigation Links
DNA Isolation From 50 to 20 mg of Paraffin-Embedded Tissue


AquaPure Genomic DNA Tissue Kit
Catalog #732-6343


Method
Sample De-paraffinization
1. Place 510 mg (0.0050.010 g) of finely minced tissue in a 1.5 ml capped tube. Add 300 l xylene and incubate 5 min with constant mixing at room temperature.

2. Centrifuge at 13,00016,000 x g for 13 min to pellet the tissue. Discard the xylene.

3. Repeat steps 1 and 2 twice (for a total of 3 xylene washes).

4. Add 300 l of 100% ethanol to the tube and incubate 5 min with constant mixing at room temperature.

5. Centrifuge at 13,000-16,000 x g for 1-3 min to pellet the tissue. Discard the ethanol.

6. Repeat steps 4 and 5 (for a total of 2 ethanol washes).

Cell Lysis
1. Add 300 l cell lysis solution, and homogenize using 3050 strokes with a microfuge-tube pestle.

2. Incubate lysate at 65C for 1560 min.

3. If maximum yield is required, 1.5 l proteinase K solution (20 mg/ml) may be added to the lysate. Mix by inverting capped tube 25 times and incubate at 55C until tissue particulates have dissolved (3 hr to overnight). If possible, invert tube periodically during the incubation.

RNase Treatment
1. Add 1.5 l RNase A solution (4 mg/ml) to the cell lysate.

2. Mix the sample by inverting the capped tube 25 times and incubate at 37C for 1560 min.

Protein Precipitation
1. Cool sample to room temperature.

2. Add 100 l protein precipitation solution to the RNase A-tre a t e d cell lysate.

3. Vortex capped tube vigorously at high speed for 20 sec to mix the protein precipitation solution uniformly with the cell lysate.

4. Centrifuge at 13,00016,000 x g for 3 min. The precipitated proteins will form a tight pellet. If the protein pellet is not visible, repeat step 2 followed by incubation on ice for 5 min, then repeat step 3.

DNA Precipitation
1. Leaving behind the precipitated protein pellet, pour the supernatant that contains the DNA into a clean 1.5 ml microfuge tube containing 300 l 100% isopropanol (2-propanol). If DNA yield is expected to be low (<1 g), add 0.5 l glycogen solution (20 mg/ml) to the isopropanol.

2. Cap the tube and mix by inverting gently 50 times.

3. Centrifuge at 13,00016,000 x g for 5 min.

4. Pour off supernatant and drain tube on clean absorbent paper. Add 300 l 70% ethanol and invert capped tube several times to wash the DNA pellet.

5. Centrifuge at 13,00016,000 x g for 1 min. Carefully pour off the ethanol. Pellet may be loose, so pour slowly and watch the pellet to ensure that it stays in the tube.

6. Invert and drain the tube on clean absorbent paper and allow to air-dry for 15 min.

DNA Hydration
1. Add 20 l DNA hydration solution (20 l will give a concentration of 100 ng/l if the total yield is 2 g DNA).

2. Allow DNA to rehydrate overnight at room temperature. Alternatively, heat at 65C for 1 hr. Tap tube periodically to aid in dispersing the DNA.

3. Store DNA at 28C.


back to top
'"/>

Source:


Page: All 1 2

Related biology technology :

1. Fast and Easy Isolation of PCR-Ready Genomic DNA from Whole Blood
2. High-Throughput Isolation of Total RNA
3. Simple Isolation of RNA from Tissue and Cultured Cells
4. Fast Isolation of Total RNA from Small Numbers of Cells
5. Comparison of Growth Techniques and Media for the Purpose of Plasmid Isolation from E. coli Using the Eppendorf Perfectprep Plasmid Mini Kit
6. Isolation of Microorganisms
7. Isolation of Genomic DNA from Saliva Using the Perfect gDNA Blood Mini Kit
8. Mouse Tail Genomic DNA Isolation Protocol(1)
9. Genomic DNA Isolation Protocol(1,2,3)
10. Basic Plasmid DNA Isolation Protocol
11. Protocol for RNA Isolation Using TRIzol Reagent with Phase Lock Gel-Heavy
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:2/23/2017)... ... February 23, 2017 , ... David Nolte, PhD ... Reception at Purdue Research Park of West Lafayette, Indiana. , The ... outstanding contributions to, and success with, commercializing discoveries from Purdue research. “This award ...
(Date:2/23/2017)... ANGELES , Feb. 23, 2017  Capricor Therapeutics, Inc. ... and other medical conditions, today announced that Linda Marbán, Ph.D, ... two upcoming investor conferences: Cowen and ... at 10:00 am ET Boston, MA ... 2017 at 9:00 am PT (12:00 pm ET) ...
(Date:2/23/2017)... Washington, USA, and CARDIFF, UK (PRWEB) , ... ... ... the international society for optics and photonics , have been named Fellows of ... individual’s significant scientific and technical contributions in the multidisciplinary fields of optics, photonics, ...
(Date:2/22/2017)... ... 2017 , ... Park Systems , a leader in Atomic Force Microscopy ... SPIE attendees and Park customers on Feb. 27, 2017 from 12-2pm at Morton’s ... Center. The luncheon will feature a talk on Automated AFM for Small-Scale and ...
Breaking Biology Technology:
(Date:2/8/2017)... Report Highlights ... The global synthetic-biology market reached nearly $3.9 billion in ... a compound annual growth rate (CAGR) of 24.0% through 2021. ... for synthetic biology. - Analyses of global market trends, with ... annual growth rates (CAGRs) through 2021. - Coverage of core ...
(Date:2/8/2017)... LONDON , Feb. 7, 2017 Report ... $12.5 billion by 2021 from $8.3 billion in 2016 ... from 2016 to 2021. Report Includes - An ... of global market trends, with data from 2015 and ... through 2021. - Segmentation of the market on the ...
(Date:2/7/2017)... Ind. , Feb. 7, 2017 Zimmer ... leader in musculoskeletal healthcare, will present at the LEERINK ... New York Palace Hotel on Wednesday, February 15, 2017 ... live webcast of the presentation can be accessed at ... replay following the conference via Zimmer Biomet,s Investor Relations ...
Breaking Biology News(10 mins):