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:8/21/2017)... (PRWEB) , ... August 21, 2017 , ... ... Healthcare Edition 2017. The awards recognize medical centers that have implemented innovative products, ... efficiency of patient care protocols, competitive advantages, financial impact/value, and market need. The ...
(Date:8/21/2017)... ... August 21, 2017 , ... Boston Strategic ... experience with Health Economics and Outcomes Research (HEOR) and ‘big data’ to provide ... US healthcare spending exceeded $3.0 trillion with nearly 1/3 spent on hospitalizations. BSP ...
(Date:8/21/2017)... (PRWEB) , ... August 21, 2017 , ... Today Aether ... a project, funded by a Bill and Melinda Gates Foundation grant, to ... Bill & Melinda Gates Foundation, also known as the Gates Foundation, is said to ...
(Date:8/18/2017)... Jose, CA (PRWEB) , ... August 18, 2017 ... ... advanced precision Lithography Equipment for the Semiconductor, MEMS, and Microfluidics Industries, announces the ... features and specifications found more often in automated production mask aligners. OAI has ...
Breaking Biology Technology:
(Date:3/30/2017)... March 30, 2017  On April 6-7, 2017, Sequencing.com ... Genome hackathon at Microsoft,s headquarters in ... will focus on developing health and wellness apps that ... Hack the Genome is the first hackathon for ... world,s largest companies in the genomics, tech and health ...
(Date:3/29/2017)... March 29, 2017  higi, the health IT company ... North America , today announced a Series ... acquisition of EveryMove. The new investment and acquisition accelerates ... tools to transform population health activities through the collection ... higi collects and secures data today on ...
(Date:3/24/2017)... 2017 The Controller General of Immigration from Maldives ... Algeen have received the prestigious international IAIR Award for the most ... Reading ... Maldives ... Abdulla Algeen (small picture on the right) have received the IAIR award ...
Breaking Biology News(10 mins):