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The following links show the statistics provided by MinKNOW for the four DNA sequencing runs I performed two weeks ago. More later…

A4_G2_DNA–report

A4_G2_DNA_run2–report

A3_G4_DNA

A1_G3_DNA–report

Another day, another DNA extraction run. This time the results were quite good, which is especially good considering that these samples were from the anemone tentacle crowns and are the samples of interest for further work. A lesser amount of tissue appears to do fine when it is liquid nitrogen ground, which is not surprising. Here are the results (Qiagen DNeasy, 12 hr digest, 50 ul elution, Qubit DNA HS assay with 3ul sample). Samples are labelled “DNA-2″. I ran a gel and the DNA quality was also excellent, some of the best I have seen. Note that realistically remaining volumes are more like 44 ul after what was used for Qubit and gel. But that still gives more than 1 ug total. Also, a second elution with 50 ul was done in a separate tube, so there is more DNA available but not quantified.

I did another round of DNA and RNA extractions yesterday. First I did some RNA extractions on some frozen tissues that consisted of only tentacle crowns, the idea being that these tissues will be the most enriched with symbionts. I used the Qiagen RNeasy kit followed by quantification using the Qubit RNA HS assay, with 1 ul of sample. Many of these preps had RNA concentrations over the detection limit so they will need to be diluted.

Note: sample G3 was extracted on 8/28/18 and quantified the same day. Also note these are labeled “RNA-2″.

Next I wanted to examine the effect of tissue concentration on DNA extractions since my previous round of extractions gave very low yields. I wondered if I had used either too much or too little tissue. These samples were ground in liquid nitrogen and I have less experience extracting DNA from such samples. It is very possible that the ground samples extract much more efficiently and that I was using too much sample. Unfortunately the results are not totally clear, as the initial quantity of ground tissue did not seem to have a major effect on DNA yield. My hypothesis is that the low quantities of tissue may have saturated the columns and therefore the higher quantities of tissue gave similar yields due to the combined effects of more DNA and yet more saturation or clogging of the membranes. The DNA was eluted with a single pass of 50 ul AE buffer and quantified using the Qubit DNA HS kit with 3 ul of sample.

Yesterday and today I did some more DNA and RNA extractions from A. elegantissima tissue using the Qiagen DNeasy and RNeasy kits. I use the Qubit DNA HS and RNA kits to assess quantity. Here are the results:

Continued RNA extractions of Anthopleura elegantissima samples using the Qiagen RNeasy kit. Further information on these samples can be found in this post. Qiashredder columns were again used for sample homogenization, and the Qubit RNA HS assay for quantification. Here are the results:

I did some further RNA extractions of Porites astreoides and Anthopleura elegantissima samples using the Qiagen RNeasy kit. Further information on these samples can be found in the previous post. I again used Qiashredder columns for sample homogenization, and use the Qubit RNA HS assay for quantification. Here are the results:

I may do some dilutions later.

I just noticed that the last time I posted was about a year ago – wow, time flies! I have primarily been working on RADseq data assemblies (i.e., being a computer jockey) and have not done really any bench work since then, hence the gap.

I’m finally getting into some RNA extractions on both coral and anemone samples. The coral samples are Porites astreoides from the Belize transplant experiment, collected in November 2016. The anemone samples are Anthopleura elegantissima from Natalie Coleman’s OA experiment last summer. Both sets of samples were flash frozen in liquid nitrogen and stored in a -80°C freezer since then. In January, I crushed the majority of these samples to a fine powder under liquid nitrogen using a mortar and pestle. The coral samples ended up being more substantial and I only needed a portion of each, and the crushed powder I obtained weighed approximately 500-700 mg per sample. The anemones samples, on the other hand, were smaller, and I only got about 60-150 mg per sample. Granted, some of the weight of the coral samples is probably skeleton.

For the extractions, I used the Qiagen RNeasy kit, with initial sample homogenization with Qiashredder columns. I used the animal tissues protocol with a final elution volume of 40 µl (single pass). The following are results from the Qubit RNA HS assay:

Overall I am pleased with these results. The only one I find odd and hard to explain is H2 End 422 – this had a relatively low amount of starting material, but I didn’t think it was that low. It may have thawed or been otherwise compromised, but I made no note of this.

1X Phosphate Buffered Saline (PBS Buffer) Recipe

Dissolve in 800ml distilled H2O:

8g of NaCl
0.2g of KCl
1.44g of Na2HPO4
0.24g of KH2PO4

Adjust pH to 7.4 with HCl.

Adjust volume to 1L with distilled H2O.

Sterilize by autoclaving.