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

 

Nucleic Acid Precipitation – 02/28/2017

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Hi all,

A little late but here is an update on the work that Bri and I worked on last Tuesday. Picking up from the chelex extraction that Carson and Allison performed and the chelex Proteinase-K extraction that Anna performed, our goal for the night was to set up and hopefully run a nucleic acid precipitation of their extractions. While we can use the DNA extracted from the chelex protocol in downstream applications like PCR, the resin in the chelex prevents accurate absorbance-based DNA quantification. Our hypothesis is that the nucleic acid precipitation would help purify the DNA and allow us to use the donated spectrophotometer we have in lab currently as a quality control check.

Background

This protocol works by relying on the fact that DNA is slightly polar. The slightly polar charge of DNA causes DNA to be less soluble in non-polar alcohol than they are in polar water. Additionally, the phosphate-sugar backbone of DNA, which is responsible for DNA’s slight negative charge, can be neutralized with the addition of salt making the DNA less hydrophilic and thus more able to precipitate.

Protocol

The protocol we used was adapted from OpenWetWare

Materials
1. 95% isopropanol
2. 3M sodium acetate pH 5.2
3. PCR grade water
4. -20C Freezer

Samples in centrifuge

Protocol
1. Weigh volume of Chelex-extracted DNA solution
2. Add 1/10th volume of 3M sodium acetate pH 5.2
3. Add 2.5 volumes of 100% ethanol
4. Place in -20 freezer overnight
5. The next day, microcentrifuge at 13,000 x g for 30mins to pellet DNA
6. Decant the supernatant
7. Air dry for 15-20mins
8. Resuspend the pellet in 50uL of PCR-quality water

Gel Electrophoresis

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Gel Electrophoresis of Sunday’s PCR Product (Tues 02.28.17)

Today we ran two gels to see what we got from Sunday’s PCR. Because we had limited time Emily Chen and I can two gels. Our sample names are based on the PCR tubes.

Made 1.5% Agarose gels (3g agarose in 200ml 1x TAE)

Add SYBRsafe to gels.

  1 2 3 4 5 6 7 8 9 10 11 12
A     x x x x x x x      
B                        
C     x x x x x x x x x  
D                        
E                        
F                        
G                        
H                        

 

I (Regina 🙂 ran a small gel with A3-A9. Emily Chen ran the larger gel with C3-C11.

We used a NEB Ready-load 100bp ladder and a Qiagen sample loading buffer. Thank-you to Riti for making the gel sheets to help with recording. (Being an idiot I forgot to log my run time. It was ~30min.)

Results

I was able to get faint smearing bands for A3-A9. The different between these samples was the annealing temperatures. The wells for this gel were very large for a small gel. I think this might be why the bands are not very crisp; however the ladder turned out great.

Emily’s gel (C3-C11) contained no bands except for the ladder (no picture). Thus primer set 3 might be the problem.

A3-A9 RW (02.28.2017)

Salmon is back and running!

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Chelex Extraction and PCR      Sunday 02.26.2017

Hi everyone,

We decided to check out protocols by doing independent runs on the weekends. Here’s my update.

Salmon DNA by Chelex Extraction

  1. Measure a .6mg piece of salmon from freezer
  2. Added to 300ul of 5% Chelex and vortexed for 30sec
  3. Incubated at 95C for 6min
  4. Vortexed
  5. Incubated at 95C for 6 min
  6. Centrifuged at 13000 for 5 min
  7. Remove and keep supernatant; discard pellet

PCR

1 rxn 36(+2 extra) rxn
ddH2O 38 ul 1444 ul
PCR Master Mix 6x (Cell&Gene) 10 ul 380 ul
Primers F&R (10ug/ml) 1 ul 38 ul (19 ul each primer)
Template DNA 1 ul 38 ul

 

PCR was run on “Sleepy” with a annealing temperature gradient of 42C-52C for columns 1-12.

Program: Main -> [B start/stop] Choose Directory -> [Down arrow -9. SoundBio, D enter] Find program -> [A list, COXX] confirm program -> [D enter] start program [D start]

 

Thermocycler – Biometra Tgradient Temp Time
Initial Denaturation 95 30(s)
Denature 95 15(s)
30 Cycles
Annealing 42-52 15(s)
Extension 72 60(s)
Final Extension 72 5(m)
Hold 4

 

Tested 3 different primer sets for the COXX gene.

  • Primer set 2 = purple
  • Primer set 3 = yellow
  • Primer set 6 = blue

Tested serial dilutions using ddH2O.

Final key:

  1. Primer set 2 – full conc.
  2. Primer set 2 – 1/10th dilution
  3. Primer set 3 – full conc.
  4. Primer set 3 – 1/10th dilution
  5. Primer set 3 – 1/100th dilution
  6. Primer set 6 – full conc.
  7. Primer set 6 – 1/10th dilution
  8. Primer set 6 – 1/100th dilution

PCR machine was set to run overnight, then moved to 4C fridge.

Diving into 2017 with NerdNite Seattle

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Welcome to 2017 our fishy friends!

Michal Galdzicki, Chinook Science Officer (CSO) of SoundBio, just presented at NerdNite in Seattle! In front of an audience of kindred nerds, our chinook swam upstage to talk about our hopes and dreams for the future of DIY salmon genotyping. We hope that 2017 will be a new run for us with an active salmon cohort, a home river in U-District, and new donations and ideas to help us build momentum. Thank to everyone that was able to attend and to NerdNite for being such an awesome venue to talk about our project!

 

2016 BioHTP Conference

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Expert writing and stellar personality skills paid off as our abstract was accepted and we presented at BioHACK THE PLANET’s conference that was held in Oakland, CA last month. You know what else paid off? Above average artistry. We were the best dressed, hands down. Watch our Chinook, Mike, give an excellent talk dressed as, well, a Chinook. What else would it be? He’s king!

Researching Different Methods

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We salmonites are continuing our search into various methods of identifying our very important SNPs. (Remember our PTC Taster lab this summer?) Some of the methods we are researching include CRISPR-Cas9, Southern Blotting, and QT-PCR. Click here for a great video of Wakanene in our garage lab offering a simplified version of what the powerful and complex CRISPR-Cas9 can do!

Summer in Seattle

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Hey everyone!

We’re back to share a brief update as summer sun in Seattle is a sacred thing. Attention has been focused on projects such as travel, kite-boarding, hiking…and organizing SoundBio workshops! Here are just a few photos from our Painting With Bacteria Workshop at Amazon’s MakerSpace. It’s always a hit and we’ll definitely do it again in the future!

 

BacteriaPaintingCat BacteriaPaintingMountains BacteriaPaintingIHeartScience 2016.08.01 Workshop1 2016.08.01 Workshop2 2016.08.01 Workshop3