Alright, so before I start getting into the juicy part of this post, I'd just like to mention that since this is lab work, there's not always a definite schedule and things are subject to change depending on whether the test subjects decide to cooperate, so, yeah...
This week, three things are planned to happen. First, we're gonna do some more diagnostic PCRs to determine the usefulness of our various buffers. Second, we're gonna isolate some Eretmocerus emiratus pupae in order to set up some special mating experiments. Third, and most tentative, we're gonna do some more microinjections of Encarsia inaron pupae.
The diagnostic PCRs, while not too tedious, are important not only for my experiment, but for all future experiments, since not all PCRs work like they should, typically as a result of which buffer is used. Today, we ran 64 different reactions with various wasp DNA samples. The difference between the reactions was the use of Gyrase-B or CO1 buffers, and the presence of BSA (bovine serum albumin). One quarter of the reactions used GyrB and BSA, another used GyrB without BSA, the third used CO1 and BSA, and the fourth used CO1 without BSA. GyrB worked best.
Oh, while I'm at it, I'll describe the ingredients of the PCR. PCR water is the solvent, buffer is used to control the pH and salt levels of the PCR ingredients, dNTPs (deoxynucleotides) are the DNA nucleotides that will make up the new DNA, primers bind at specific locations on the template DNA (this is where the DNA replication starts during the PCR process), Taq (from the bacterium Thermus aquaticus) elongates the dNTP strands, Magnesium Chloride affects the binding stringency of the primers, and BSA sometimes (but not always) improves the performance of the PCR reactions.
So, we're going to isolate more E. emiratus pupae later this week (Wednesday or Thursday), but the adults won't be ready to use until Monday. We're planning on using this new generation of test subjects to perform some competitive mating experiments. We're going to place a W- female and 2 males of differing symbiont infection statuses to determine which, if any, the female prefers. It'll be quite interesting if somehow she can tell that the W+ male is no good for her.
Finally, maybe on Friday, I'll get to do some more microinjection. It's an excessively difficult process, though, so I won't be getting my hopes up for too much success. Anyway, microinjection is done by placing a series of E. inaron pupae of alternating symbiont infection status on a slide and sucking the juices out of one pupa and putting them into another pupa, hopefully transferring the first wasp's infection. The pupae need to be juicy for this to work, and even then, success is not guaranteed, and then needle breaks easily.
Next update on Friday!!!
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