Monthly Archives: August 2018

DNA extraction – A. elegantissima

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.

Sample Vol ul DNA ng/ul
G4 50 37.3
A3-2 50 36.7
A1 50 too high
A1-2 50 too high
A4 50 31.5
A3 50 36
G2 50 28.6

Ae_DNA_082118 copy

More anemone nucleic acids

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″.

Sample Vol ul RNA ng/ul
A1 40 93
A3 40 too high
A4 40 190
G1 40 too high
G2 40 too high
G4 40 172
G3 40 46

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.

Sample Vol ul DNA ng/ul
A4-L 50 29.5
A4-M 50 28.6
A4-H 50 26.3

DNA and RNA extractions

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:

Sample DNA ul DNA ng/ul RNA ul RNA ng/ul
A1 50 9.34 40 172
A2 50 7.72 40 182
A3 50 6.17 40 69
A4 50 5.85 40 200
G1 50 8.51 40 88
G1 50 6.89 40 82
G3 50 7.64 40 104
G4 50 6.06 40 28