Tag Archives: Axa

DNA Isolation – Geoduck

Isolated additional geoduck gDNA from the two fresh (now frozen) geoduck’s that Brent provided me with on 20140212 so that we can potentially isolate RNA from the same geoducks to tie in with the DNA Illumina sequencing that Axa will be conducting. Isolated DNA using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol (incubated minced siphon tissue at 56C for 3hrs). Eluted with 75uL of ddH2O and spec’d on NanoDrop1000.

Note: Initial specs were too low for Axa’s requirements (50uL, >= 500ng/uL). SpeedVac’d samples to concentrate, brought volume to 55uL and then spec’d on NanoDrop1000.

Results:

Samples look good. Will send Axa 50uL of all samples, excluding GD01 since that sample is below his desired concentration AND I believe he probably doesn’t want to wait for this DNA any longer.

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DNA Precipitation – Geoduck DNA from 20140213

After speaking with Axa regarding the DNA concentrations, he would like the DNA from the ethanol-fixed tissue to be more concentrated, and he wants them in ddH2O instead of Buffer AE (from the Qiagen DNeasy Kit). So, I preformed a standard ethanol precipitation. Added 0.1 volumes of 3M sodium acetate (pH = 5.2) [15uL], 2.5 volumes of 100% ethanol [412.5uL] and incubated @ -20C over the weekend.

Pelleted DNA by spinning 16,000g, 15mins @ 4C. Discarded supe, washed pellets with 1mL 70% ethanol and re-pelleted the DNA. Performed a second wash with 70% EtOH, pelleted DNA, discarded supe, resuspended DNA in 75uL of NanoPure H2O, and spec’d on NanoDrop1000.

Results:

Will spec when I re-isolate DNA from “fresh” geoduck samples for coordinating Axa’s DNA sequencing project with our potential RNA-seq project. See 20140219.

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DNA Isolation – Geoduck

Since yesterday’s DNA isolation failed to yield sufficient quantity of DNA from the ethanol-fixed samples, I isolated additional DNA from the same samples.

Isolated gDNA from 6 geoduck siphon samples provided by Brent using the Qiagen DNeasy Spin Kit. Samples were as follows:

  • Langley 006
  • Langley 007
  • Langley 008
  • Langley 009

The Langley samples were fixed in ethanol in 2006. Geoduck 01/02 were fresh geoduck siphon samples, taken from two live, juvenile geoduck (the rest of the bodies were frozen at -80C).

The samples were processed according to the Qiagen protocol (but utilized an overnight incubation at 56C with Proteinase K), eluted in 50uL of Buffer AE, combined with the corresponding DNA from yesterday, mixed throughly and spec’d on a NanoDrop1000.

Results:

Now have sufficient quantity of DNA for all four of these samples. Will contact Axa (the person who this DNA is intended for) to see if he requires a specific concentration/volume.

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DNA Isolation – Geoduck

Isolated gDNA from 6 geoduck siphon samples provided by Brent using the Qiagen DNeasy Spin Kit. Samples were as follows:

  • Langley 006
  • Langley 007
  • Langley 008
  • Langley 009
  • Geoduck 01
  • Geoduck 02

The Langley samples were fixed in ethanol in 2006. Geoduck 01/02 were fresh geoduck siphon samples, taken from two live, juvenile geoduck (the rest of the bodies were frozen at -80C).

The samples were processed according to the Qiagen protocol (but utilized an overnight incubation at 56C with Proteinase K), eluted in 100uL of Buffer AE and spec’d on a NanoDrop1000.

Results:

The person who needs these samples (Axa) needs at least 25ug of DNA. The two fresh samples (Geoduck 01 and Geoduck 02) yielded more than sufficient quantities of DNA. The Langley (i.e. ethanol-fixed) samples did not yield sufficient DNA and I will need to isolate additional DNA from these samples.

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