Yesterday I did a second trial run with the same P. astreoides samples used in the previous run (sample #s 1, 10, 97, 100; see previous post). This time, however, I used the Qiagen DNeasy Blood and Tissue Kit. I used approximately the same amount of tissue used in the previous runs, perhaps slightly more. All tissue samples were largely detached (sloughed off) from the skeleton as before, so for most samples I simply scooped loose tissue from the tubes. I did not do any homogenization. However, I did rinse the tissues three times in PBS, compared to only once in the previous run with the EZNA Mollusc Kit. Another thing that I did differently was a shorter 2.5 hr lysis at 56ºC, instead of a 24 hr lysis at 37ºC with the mollusc kit. I chose this lysis duration mainly out of convenience since I was unable to come back the following day. I also vortexed the samples periodically during lysis. At the end of the lysis period, I noted the following with respect to the appearance of the samples: samples 1 and 10, no visible skeletal material, some bits of intact tissue; sample 97, some skeletal material present, very little visible tissue; sample 100, no skeletal material, many small bits of tissue. So from these I estimated that the only sample that was fully lysed was #97. However, I think this was also the sample with the least amount of tissue.

The good news is that this time there was significantly more DNA in most of the samples. Here is the nanodrop report (the second half of the samples with a -2 are the second elution).

 

My sense is that sample 97 had low DNA concentration due to a low tissue quantity. As for sample 100, I think there may have been too much tissue and incomplete lysis. But I would not say there was huge variance in the amount of sample used, so the large differences in DNA quantity are a bit perplexing. In any case, the results are encouraging and I will move forward with DNeasy for all samples, perhaps with a longer lysis duration and some light homogenization.