User:Anthony Salvagno/Notebook/Research/2009/02/09/Isolating DNA

Today we focused on steps 16-23 (end) of the protocol. Once again there were some minor changes. There were also some further and more in depth descriptions.

The Expanded Protocol

1. spin 10 min
   • We got to use the big centrifuge. That was pretty cool.
   • Spin at 10k.
2. resuspend pellets in 500uL 1mL TE
   • Basically I got rid of all the EtOH and then added TE to it. We had a much larger pellet than anticipated so we doubled the amount of TE to use.
3. transfer to 2mL eppi
4. add RNaseA .1mg/mL
   • Kelly wasn't sure how much to add so this was previously left blank in the protocol. Updated now (not on protocol page though).
5. incubate 37C for 30 min
6. extract with Phenol/Chloroform
   • because we had a large yield in terms of DNA (I feel like a farmer, but a cool futuristic DNA farmer) we needed to alter a few things.
     1. separate the eppi tube samples (4 up to here) in half (making 8 tubes)
     2. add equal volume of Phenol/Chloroform (P/C) to each tube
        • we had approx 1.5mL of DNA/stuff in each tube so put 750uL (.75mL) into tubes. Then we added 750uL of P/C
     3. create immulsion using the vortexer
        • This comes from the fact that the solutions we are combining aren't mixable (soluble?) and so when we vortex them we just get a foamy solution that will dissociate in time.
     4. Spin 10 min at highest speed (13k?)
     5. Pipette top layer of solution (should be DNA) and recombine halves
        • In the tube you can see 3 layers. The top is the DNA, the middle is a super thin layer of lipids and gunk that we don't need, and the rest is the P/C plus whatever dissolved in it.
        • Once you get most of the DNA out, it becomes hard to pull out more DNA. The lipid layer and P/C layer collapses on the DNA layer and forms a small bubble. The lipids then start to go back into the DNA solution. At this point it is worthless to salvage any more DNA... poor little guys.
     6. dispose of P/C properly.
7. EtOH precipitate DNA (>1hr)
   • another stable stopping point, but this time the show must go on.
8. DNA purification steps:
   1. put 1/3 of DNA solution into 2mL eppi tube (we used 1/3 since there was about 4mL of total solution)
   2. spin tube for 5 min at highest speed in cold room (this is 15k)
   3. pipette out all but DNA pellet
   4. add next 1/3 of original solution to the DNA pellet tube (technically you will have 2/3 of the DNA in 1/3 the volume)
   5. repeat steps 2 and 3
   6. add final 1/3 solution to tube (all DNA should be in there now)
   7. repeat steps 2 and 3
9. dissolve DNA in 50-100uL TE
   • we used 100uL amount because Kelly felt as if we had a lot of DNA to work with.
   • at this point we combined all 4 of our DNA tubes (performing DNA purification steps for all 4 tubes) into one tube labeled w303a GDNA with the date (2/9/09)
10. measure DNA concentration on nanodrop
    • Kelly figured that we had too much DNA and so made some dilutions for the nanodrop to be able to read accurately. We found out that it wasn't as much as we thought it was, but it was still more than we needed.
    • nanodrop readout was: 213.8ng/uL -> 85.52ug of DNA
    • Kelly said we only needed about 20ug
    • Steve Koch 17:24, 10 February 2009 (EST)I changed above to micrograms while

Comments and Other Notes

• I don't like trying to suck out needed liquids from a dissociated solution. There has to be a microfluidics setup to effectively pull out only needed layers.
  • Steve Koch 01:09, 10 February 2009 (EST): I couldn't agree with you more. The inexactness of phenol chloroform extraction and trying to pipet off a supernatant without disturbing the pellet drive me crazy and stress me out like crazy.
• Someone on friendfeed pointed out that there would be a lot of walking in my new deployment. Boy were they right. Kelly and I go back and forth between labs and across the hall very often. There is almost no sitting time. I don't mind at all though.
• Today was the most active day since I've been here (in terms of walking between labs and stuff) and so I walked by way more people then I usually do. At CHTM as I walk by people I always stare them down (non-menacingly) because I am awkward like that. I find that other scientists will either do the same (so we both awkwardly make eye contact without acknowledging each other) or one/both of us will try and pretend we are looking at something important to avoid eye contact. At Cancer Research however, I feel like people here are generally more friendly and less awkward. I smile and say hi to almost everyone (even people I don't know) and it helps that I am trying to be more open. Although I did pass a couple people who purposely looked around the hall to completely avoid eye contact. One dude even continuously rolled his eyes around. It was trippy.
  • Steve Koch 01:09, 10 February 2009 (EST): I am a total head-nodder. There's one guy at CHTM whose name I don't know but we've head nodded like 1,000 times since I joined UNM. Plus I even say hi to Larry's girlfriend's dad all the time.