Genomic extraction using Tail Lysis Buffer — 03/07/2017

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For this week’s Salmon Night we picked up on the nucleic acid precipitation protocol we began last week and explored another technique for genomic DNA extraction.

Background

Genomic DNA extraction from tissue has three main steps. The first step is typically disrupting the tissue and breaking open the membrane of cells. The second step is the removal of containments and unwanted cellular material (typically proteins and lipids). And finally, recovering and purifying the DNA from solution.

In the Chelex extraction procedure, the chelex resin themselves, along with boiling, help disrupt the tissue. The chelex resin also acts via ion exchange to trap Mg++ ions in the sample. Mg++ is a necessary cofactor for DNAases, or enzymes that degrade DNA, so by removing them from the solution, potential DNAases are inactivated. Centrifugation is then used to clarify the solution. Here is the protocol we have been using.

Other methods for extraction include a using a combination of a detergent, a proteinase, a chelating agent, nonpolar alcohols and salts. The detergent helps remove lipids (think oils), the proteinase helps degrade unwanted protein contaminants, the chelating agent traps the DNAase cofactor Mg++ and the nonpolar alcohols and salts help precipitate, and thereby purifiy, the DNA after centrifugation.

Protocol

Solution after step 1

To make the genomic DNA extraction solution we followed the recipe made by a mouse genotyping lab (Tsai Lab in the links below)

Tail Lysis Buffer

50 ml  1 M Tris.Cl pH8.0
5 ml     500 mM EDTA pH8.0
10 ml  10% SDS
20 ml  5 M NaCl

1. On 03/06/2017, Zach and I weighed out approx 200mg of Salmon into a 1.7mL Eppendorf tube and added 500uL of the Tail Lysis buffer along with 7.5

Solution after step 2

20mg/mL Proteinase K. We set the solution to rock overnight at 55C.

 

2. Centrifuged the samples at 13,000 rcf for 10 mins

3. Added 500uL of 91% isopropanol and inverted 3-5 times

4. Incubated the sample and the centrifuge motor at -20C for 20mins

5. Decanted the supernatant (being mindful of the DNA pellet)

6. Washed the pellet with 70% ethanol

DNA pellet after step 7

7. Decanted the supernatant (being mindful of the DNA pellet)

8. Let the pellet air-dry for 20mins

9. Resuspended the pellet in 20uL sterile water.

 

 

Sources:

Tsai Lab -http://tsailaboratory.mit.edu/about/protocols/preparation-of-genomic-dna-for-genotyping/

Carlton Science Education and Resource page – http://serc.carleton.edu/microbelife/research_methods/genomics/dnaext.html

BioTechniques Journal – http://www.biotechniques.com/BiotechniquesJournal/2013/March/Chelex-100-as-a-Medium-for-Simple-Extraction-of-DNA-for-PCR-Based-Typing-from-Forensic-Material/biotechniques-341022.html