Friday, February 20, 2015

2 20 2015 Test DNA Extraction Methods Part 2

Yesterday, after discussing the poor results from the 96 well plate extraction, I decided to directly compare the Qiagen DNEasy kit with the DNAzol method. You can see what I did in yesterday's blogpost. Today I completed the DNEasy extraction with only an overnight incubation instead of 24 hours due to concerns of dnases destroying genomic DNA during the incubation. I started the lysis incubation yesterday.

Today I performed the following protocol for DNEasy extraction.

  1. Vortexed Samples to mix up lysed tissue
  2. Allowed them to sit for 10 minutes while I made up a gel. 
  3. Added 200 ul Buffer AL with EtOH from the 96 Well Kit
  4. Added 200 ul 100% EtOH
    1. This was a mistake as the 96 well kit premixes them and the singles kit does not. We ended up using a 75% EtOH, 25% Buffer AL Solution. 
  5. Vortexed thoroughly
  6. Pipetted into a column
  7. Centrifuged at 6000 g for 1 minute
  8. Discarded collection tube
  9. Added 500 ul AW1 for wash and new collection tube
  10. Centrifuge at 6000 g for 1 minute
  11. Discarded collection tube
  12. Added 500 ul AW2 for wash and new collection tube
  13. Centrifuged at 10,000 g for 6 minutes
  14. Discarded collection tube
  15. Placed column in labelled 1.5 ml tube
  16. Added 200 ul AE elution to column
  17. Incubated 1 minute at room temp
  18. Centrifuged at 6000 g for 1 minute
  19. Repeated steps 16, 17, 18. 
  20. Discarded column and stored at room temp. 
Following the completion of the DNeasy extraction I ran a gel with both sets of isolations. 

0.9% Gel:
50 ml 1X Low TAE
0.45 g Agarose

Microwaved gel for 4 minutes. Allowed to cool for 10 after thoroughly dissolved. Poured gel smoothly. Allowed to set for 30-45 minutes. 

Loaded wells with 
10 ul 100 bp Ladder
25 ul DNA with 2.5 ul Loading dye. 

Wells organized like:

SampleLadder1N9-12 181N9-12 191N9-12 201N9-12 211N9-12 181N9-12 191N9-12 201N9-12 21EmptyLadderEmpty

Gel comparing DNAzol to DNEasy
First, There does appear to be intact High Molecular Weight DNA. Second, surprisingly the Qiagen produced a higher yield in the same sample. This makes me wonder if the 24 hour incubation allowed more DNases to chew through the DNA. If I do the plate extraction again, I will only allow for a 12-18 hour incubation period. Also I think the smaller tissue size helped immensely. It look like high molecular weight does vary between samples with less of it in two of the samples. Since all samples were processed the same way I'm not sure what this could mean in terms of sample quality and yield for sequencing. 

Moving forward, we should consider doing the 96 well extraction again with smaller tissue sizes, less incubation time, and possible the 75/25 mix of the AL solution.

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