Thursday, February 12, 2015

2 12 2015 DNA Isolation Part 2

As with yesterday's post, I'm finishing up the 96 Well Plate Qiagen DNEasy extraction kit. Today I worked in the MERLab because of the proximity to the centrifuge that can fit the kit plates. I brought all the reagents, pipetters, pipette tips, and gloves. I ended up borrowing a graduate cylinder but I cleaned it thoroughly before and after use. A couple things to note, I lost some of the lysate from the samples as a couple of the caps were not as secure as I thought were and popped open during the first shaking step. Luckily these mostly came from the first column which as you can tell by the plate set up only affected one or two samples from each population sampled. Also on the elution step I repeated it twice because on the first run through, Row A received half as much buffer as intended. When I repeated the elution I over filled Row A and now there is about 500 ul of isolate instead of 400 ul. I will run a gel on the samples next week to check quality. Hopefully Row A is not extremely diluted. Though I can probably concentrate it without too much trouble. Also I used the sample channel reservoir for every solution. Between solutions I rinsed with water and did a final rinse with DI Water. I wiped it dry with a paper towel.

Today's Procedure:

  1. After incubating roughly 24 hours at 56 C samples were sealed and shaken (lost some lysate due to poor seals on a few caps).
  2. Centrifuge at 5700 rpm for 20 second.
  3. Uncapped/Added 410 ul premade Buffer AL/EtOH solution.
  4. Capped with new caps. Shook for 20s.
  5. Centrifuge at 5700 rpm for 20 second.
  6. Placed a DNeasy 96 well column plate on the previously used S Block. 
  7. Attempted to remove 600 ul of lysate from each sample
    1. Some had more some had less due to volume of tissue and loss of lysate. Most sample tubes appeared visually to be at roughly the same volume.
  8. Sealed plate with Airpore sheet.
  9. Centrifuged at 5700 rpm for 11 minutes.
  10. Decanted solute from S Block. Wiped with Paper towel. 
  11. Removed Airpore tape. 
  12. Added 500 ul Buffer AW1 to each sample
  13. Seal with Airpore tape.
  14. Centrifugee at 5700 rpm for 6 minutes. 
  15. Removed Airpore tape. 
  16. Added 510 ul Buffer AW2 to each sample. 
  17. Centrifuged at 5700 rpm for 16 minutes. (Used no airpore tape per instructions)
  18. Moved DNeasy 96 well column plate to Elution Microtubes plate. 
  19. Added 200 ul Buffer AE to each sample (Row A got ~100 ul)
  20. Sealed with Airpore tape. 
  21. Incubated for 1 minute at room temperature. 
  22. Centrifuged at 5700 rpm for 3 minutes. 
  23. Removed Airpore Tape. 
  24. Added 200 ul Buffer AE to each sample (Row A got 400 ul)
  25. Sealed with Airpore Tape
  26. Incubated for 1 minute at room temperature. 
  27. Centrifuged at 5700 rpm for 3 minutes. 
  28. Removed DNeasy column block from Elution Microtubes plate. 
  29. Sealed Microtubes with rubber tops flipping which which side the openning tab was on for every column. 
  30. Labelled sample with "O.lurida sample processed 2/12/2015. 09/14 Oyster, 10/14 Fidalgo Manchester. JH"
  31. Stored in the 4 C in 213. 

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