Wednesday, March 26, 2014

Plasmid Prep

This week I have been working on generating plasmids in trusty Escherichia coli, aka E. coli. A plasmid is a small circular, replicating piece of DNA. For research, we can put any number of genes in the plasmid and whatever we transform will then make them! Right now I am simply learning the method so my plasmid is generic pCambia 1304, yawn. The nice thing about this plasmid is it has two reporter genes (GFP & GUS) that are on all the time in the cell. So any cell expressing the plasmid will either glow green under UV light (GFP) or fluoresce in a special assay (GUS) making them easy to identify. 

Monday, I put the plasmid into the E. coli cells, it was really easy as we were using special E. coli that is ready to accept new DNA.Then I had to let them be fruitful and multiply.. in a very smelly media. Seriously the media smells like warm trash.. then you let E. coli grow in it for a while and it REALLY smells. Today was a smelly, smelly day! BUT I had a lot of E. coli that would all have copies of my plasmid inside of them... next step, get those suckers out!

That is what I spent all day working on, and I'm still not done! Thankfully the procedure has a few "holding pattern" points where it can run for X minutes to overnight. I stopped at the last holding spot and was the last one out of the lab. Which is weird for me as I'm usually the first to leave every day to get Boo from daycare. But this week he is with his Dad (more on Friday about that), so I could stay late.

Isolating plasmids is a lot more complicated than you might think. Getting DNA is easy, I can literally do it with dish soap, salt water, Everclear, and a strawberry in my kitchen. Plasmids though, they are a different story. Inside every E. coli cell is the cells natural DNA, the long strand of genomic DNA which is 4.6 million base pairs long. I then add a plasmid that is 6128 base pairs long. That is a lot of DNA floating inside this tiny E. coli cell.. and I only want the plasmid! The genomic DNA is actually a big pain in my booty. Thankfully, despite the fact that they are made of the same material, there is a way to separate them.. any guesses about what we use to do that?

Size. Remember genomic is 4.6 MILLION base pairs.. that's a LOT bigger than my plasmid. Perrrrfect! Using size to your advantage is great, but it takes a lot more time and care than your standard crack the cells and rip the DNA out extraction method. If you break that large genomic DNA strand into little ones it will end up in the fraction that you think is all plasmids. The bacteria must be carefully open and all the other macromolecules (fats, carbs, DNA, RNA, etc.) removed with a series of chemical and centrifugation steps.

For this procedure, the centrifuge is the key. Different speeds will isolate out different sized particles. A nice "slow" speed will get all the big stuff, like unbroken E. coli, large chunks of membranes etc. A fast spin will bring down the smaller molecules. By doing a bunch of purification and centrifugation steps, you can slowly get rid of the big stuff and end up with just the little plasmids. Tonight, while I sleep my little plasmids should be precipitating out in their lovely alcohol bath in the freezer. Tomorrow I will wash them and quantify them, hopefully there will be a LOT cause I need a ton for step #2.. transform some Arabidopsis protoplasts :) Hopefully in a future post I'll have pics of some beautiful glowing protoplasts that I made.

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