Grad Diary 3/22/10

For the past three weeks or so I have been unsuccessfully trying to insert a particular sequence into a plasmid vector containing the HPV16 genome. What makes this particular cloning experiment hard is that the vector is rather large (~10kb!) and my insert is not only small, but it is a mixture of various polymers of the same sequence (anywhere from five to over a hundred repeats). I don't know how many agar plates I went through (maybe twenty?) while I've been trying to get things to work. Jian has been helping me troubleshoot throughout the way to optimize the ligation reaction, DNA extraction methods (for example, is it better to do a gel extraction or the tried and true phenol:chloroform:isoamyl alochol extraction with ethanol precipitation?) It got to the point where I am literally running out of both vector and insert.

Finally, after working on this for weeks, I came in this morning and checked my plates and was surprised to see an explosion of growth! Unfortunately, even my ligation control plate (which is vector without insert) also showed an explosion of growth, so that means that one of the key steps that has been hampering my progress is the de-phosphorylation step with CIP. We ordered new CIP. Fortunately, this is still pretty good since, at first blush, it seems like my ligation reactions still had more growth than the controls. I re-plated my reactions at a much smaller volume, and hopefully they will have reasonably sized colonies for me tomorrow.

It was one of those moments where, after weeks of banging my head against the wall, there was suddenly an "Aha!" moment, and things looked like they are finally falling in place. There is still some work to do to see if there is anything usable, but even if I have to start over, I now have everything in place that it will only take a week to go from start to finish, instead of almost a month. Still, Jian and I have decided that I will stop after this week so I can focus more on reading and preparing my ideas for my thesis project(s). Maybe I can get one of the undergrads to finish things for me. It's kind of nice being able to say that, ha ha.