We made our first try at extrtacting and amplifying DNA from kleptoplasts.  It was relatively straightforward to get the tissue from the slug.  First, she was “relaxed” with isotonic MgCl2, which blocked synaptic transmission and paralyzed and anesthetized her.

Elysia clarki relaxed with 400 mM MgCl2. 4/27/16

Then, a small piece of parapodium was removed (see her left side, at bottom).  DNA was extracted using the same method as for the plants.

Elysia clarki with piece of parapodium removed for DNA extraction. 4/17/16

In case you were worried, she was fine the next day.

Elysia clarki one day after piece of parapodium removed. Hungry and looking for food. 4/17/16

We also tried a new species of algae, which I am calling Avrainvillea.  It may be Rhiphilia, I’m not completely certain.

Alga extracted 4/27/16. Probably Avrainvillea.

Unfortunately, the amplification was not a success this time around.  The positive control samples, DNA extracted from Bryopsis during previous sessions, worked well.  “Avrainvillea” showed a much weaker signal, and there was no signal from the slug extract.

Results from week of 4/25/16

A number of things may have gone wrong.  The slug sample was extremely slimy with mucus, and those polysaccharides could have interfered with extraction or amplification.  Also, the Bryopsis-specific primers may not have annealed adequately with the DNA from Avrainvillea and whatever is in the slug.

Now that things are calming down after the semester, I am going to try again, making a few changes.  Most importantly, I have ordered “degenerate” primers, which contain a mix of sequences that will complement just about any algal sequence.  Still waiting on tips regarding the slime.