I have been moving forward with extraction of DNA from P. astreoides samples using the Qiagen DNeasy kit. On Monday/Tuesday I did 16 samples, then another 10 samples (actually 9; one was divided in two). For the first set of 16, I did an overnight lysis (6pm to 9am) with no homogenization. Most of the tissue was very loose and easily scraped off the skeleton or scooped from the bottom of the tube. My initial results (see previous post) suggested that larger quantities of tissue were more successful, so I was fairly generous with tissue quantity. I did two 200 ul elutions into two separate tubes. I measured DNA quantity in the first elution with the Qubit HS assay. Here are the results (again, for the first elution only):
| Sample # | Qubit conc. (ng/ul) | Sample volume | Total DNA quantity (ng) |
| 15 | 5.92 | 200 | 1184 |
| 16 | 3.46 | 200 | 692 |
| 96 | 6.82 | 200 | 1364 |
| 19 | 5.4 | 200 | 1080 |
| 14 | 3.92 | 200 | 784 |
| 82 | 7.38 | 200 | 1476 |
| 98 | 7.92 | 200 | 1584 |
| 13 | 10.4 | 200 | 2080 |
| 12 | 3.1 | 200 | 620 |
| 18 | 5.88 | 200 | 1176 |
| 99 | 5.16 | 200 | 1032 |
| 17 | 10 | 200 | 2000 |
| 95 | 5.88 | 200 | 1176 |
| 11 | 10.4 | 200 | 2080 |
| 80 | 10.8 | 200 | 2160 |
| 81 | 8.82 | 200 | 1764 |
For the next set of 10 samples, I did a few things differently. Again, I tried to be generous with tissue quantity. For a few samples, the tissue volume of the pellet was nearly 100 ul. I ranked the tissue quantity on a scale of 1 to 10, 1 being the lowest. I also ranked the sample color after the lysis period to get a second qualitative measure of tissue quantity. I homogenized three of the most dense samples lightly with a plastic pestle (86, 90, 83). The lysis period was from 4:30pm to 9:30am. The lysis did not appear complete, so for the last hour of lysis, I added an additional 180 ul of buffer and 40 ul of proteinase K, and disrupted a few of the most dense samples by pipetting. Sam suggested elution with 50 ul instead of 200 ul, and reusing the original eluate for the second elution. I quantitated the DNA with both Nanodrop and Qubit for comparison. Here are the results:
| Sample # | Tissue quantity rank (1 = low) | Color rank after lysis | Nanodrop DNA conc. (ng/ul) | Qubit conc. (ng/ul) | Sample volume | Total DNA quantity (ng) |
| 87 | 1 | 1 | 166.2 | 45.6 | 50 | 2280 |
| 89 | 2 | 3 | 122.9 | 48 | 50 | 2400 |
| 85 | 3 | 4 | 313.9 | 143 | 50 | 7150 |
| 88b | 4 | 2 | 214.2 | 121 | 50 | 6050 |
| 84 | 5 | 6 | 398.1 | 188 | 50 | 9400 |
| 91 | 6 | 9 | 329.7 | 194 | 50 | 9700 |
| 88a | 7 | 5 | 292.6 | 182 | 50 | 9100 |
| 86 | 8 | 7 | 359.4 | 234 | 50 | 11700 |
| 90 | 9 | 8 | 312.3 | 218 | 50 | 10900 |
| 83 | 10 | 10 | 377.7 | 194 | 50 | 9700 |
The Nanodrop quantity estimates were approximately double those from the Qubit. Qubit values were used for estimation of total yield in the last column. Also, from my qualitative estimate of tissue quantity, it appears that higher quantities tended to be better. If you plot the data, there appears to be an asymptote, so I may have reached optimum tissue quantities.
