Week 7

I got right back into the swing of things this week, and learned how to perform even more procedures as my project progresses.

On Monday, my mentor told me that he had already gone ahead and sent in the previous week's samples for sequencing (the final step of the cloning process) during my hiatus. The results hadn't arrived yet, though, so I instead practiced the next step: in vitro transcription.

Although DNA is the ultimate blueprint for making proteins and therefore organisms, it is not actually directly translated into them. That role belongs to an intermediate molecule known as RNA, which is produced in the cell's nucleus using DNA as a template. After RNA is translated into proteins, it breaks down, halting the production of proteins but leaving the original DNA unaffected.

What we had at that point was DNA; injecting it into the egg cells would do nothing, as the cell would continue to only express the DNA in its original genome. However, RNA would be immediately translated to the proteins that we desired. So the next move was to synthesize RNA from DNA (i.e. transcription), but not in a cell-- in the lab (i.e. in vitro).

Whew. I'll try not to do that again.

Although RNA synthesis is a very different procedure than cloning, it does use some of the same machinery-- namely, a PCR thermocycler and my old friend gel electrophoresis. I spent most of Monday and Tuesday doing a "practice run" of the entire method.

On Thursday, the sequencing results finally arrived-- with a mixed outcome. Two of the four DNA sequences we had been trying to produce were verified, but the other two were not, possibly due to a low yield. Dr. Chang told me it was okay to proceed regardless, as they had all still been verified by gel electrophoresis.

So I then started the transcription procedure for real. On Friday, though, I already encountered an error; the very first PCR step wasn't confirmed by a gel I ran, for some reason. I'll try again using a different PCR machine on Monday, and it'll hopefully work then. If not, I might have to go through FastCloning again, so keep your fingers crossed!

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