What's up graduate student office?

I grew up liquid bacteria cultures last night so I could harvest their plasmids this morning, but alas, my liquid cultures didn't grow overnight. They looked this morning pretty much like they did last night:

I grew four different cultures last night from the same bacteria colony. This colony I has semi-recently used for a plasmid harvest (plasmid prep), so I was shocked to see they hadn't grown. On the left (the darker cultures) is 2xYT buffer (2 times the amount of Yeast Extract Tryptone), an especially nutrient-rich media, and on the right (the lighter cultures) is the standard LB broth (which I just learned from Wikipedia is commonly incorrectly called Luria broth). I set up one of each culture at 1 volume and 2 volumes of the antibiotic ampicillin. I usually grow my cultures at 2 volumes (100µg of amp per milliliter). The amount of antibiotic is very important--let me explain: we're growing these bacteria for their plasmids (sort of like growing people for organs, like in the movie The Island--sorry if I spoiled that one for you hahahaha), and the plasmids have a gene for antibiotic resistance in addition to the other parts of DNA we're cloning the plasmids for (click here for a 101 post on plasmids). Therefore, any bacteria that have a plasmid should be able to survive in the presence of ampicillin. This means we need to add ampicillin to our cultures, to weed out the bacteria that don't have the plasmid. However, if we add too little ampicillin, some bacteria that don't have the resistance gene (from the plasmid) may still be able to survive. If we add too much ampicillin, none of the cells may survive. It's a catch-22: too many bacteria cells (especially those without the plasmid) due to no or too little ampicillin, will give us poor plasmid harvest yields. (This is particularly problematic because bacteria without the plasmid may end up growing faster than counterparts with plasmids, because they don't have to invest the energy into making the plasmids.) However on the other end, too much ampicillin will prevent the growth of cells and I won't get any cells to harvest their plasmids.

In my case, my bacteria colonies were probably too old to start new liquid cultures from. Bacteria colonies are best used if they are actively growing, because they are their healthiest at that point. We'll have to see if I can get these current colonies to grow anymore.

In other news, I'm using my new graduate student desk (which I'm sharing with my labmate Jessica):

It's pretty cool to have a space in the graduate student office, because now I finally feel like a graduate student at Clark. Just settling in as you can see. Jess and I will be using this space primarily to do our reading and writing, but it could also be used to plan out experiments, do research, etc.

Science is still not being linear (enhanced with video!)

So science is still not being linear for me, as I continue to have problems with my PCReactions.



I ran the gel this morning, and got this:

Lanes: 1 - 1 kb ladder; 2-5 - PCR reactions; 6 - positive control; 7 - negative control; 8 - 50 bp ladder

Aaaaaand I got nothing, beside the positive control (lane 6, 2 lanes above the bottom ladder).

Sometimes science isn't linear (and I'm not talking about exponential growth)

Sometimes science (my project) isn't linear, and I've been getting caught up in this recently with my posting.

I wanted to do a series of posts and videos on the process of transformation and the completion of my first NiR plasmid, but what I found was that sometimes things don't work out the way you want.

So, let me backtrack a bit.

As I mentioned before, it appears I have transformed bacteria colonies that have my PCR insert. Great! But I've been having trouble getting the insert to amplify out of the plasmid once again. I grew up several bacteria colonies that looked like they had my insert (white colonies on X-gal) and performed a plasmid prep that yielded very little plasmid DNA. I need a decent amount of this plasmid to allow me to digest (cut) out and obtain the insert.

Now, I'm trying to do yet another PCR reaction in a much larger volume (50µl rather than 10-20µl) using the plasmids I obtained from my lame plasmid prep. If this works, I'll have a lot of copies of the NiR terminator insert, which I can then slice off the ends with restriction enzymes. Then, the insert would be ready for the next step.

That is, if I can get this to work. :-\

It's Transformation Time.

Last time I checked in, my PCR reactions that were supposed to add restriction sites to either end of the nitrite reductase (NiR) terminator region appeared to work and work well. When I ran the PCR reactions on a gel, the amplified DNA bands were really strong and were the correct length.

This gave me the go ahead to continue the path in isolating & altering the NiR terminator in order to yield a sequence of DNA to fit the final NiR plasmid for my project's experiments.

Now I need to insert the NiR terminator into a vector plasmid, transform it into bacteria, and digest the NiR terminator back out of the plasmid to double check that the terminator I got on the gel from my PCR reaction (above) is the correct piece of DNA before I insert it into the NiR plasmid to complete the final NiR plasmid.

We use bacteria to amplify pieces of DNA because of their quick generation times. If you insert a plasmid into bacteria, they will duplicate the plasmid as if it were their own DNA as they grow and divide. A plasmid is a ring of DNA, which is essential for this to work, because bacteria will cut up and destroy any loose pieces of linear DNA. In order to get our NiR terminator to be duplicated by the bacteria, we insert it into a vector plasmid first. The vector plasmid is designed to accept small pieces of DNA from PCR reactions, lock in that piece of DNA within the plasmid. This plasmid can then be transformed into bacteria.

Bacterial transformation is really easy. Once the vector plasmid complete with our PCR DNA is ready, we add the plasmids to specially-altered E. coli cells, incubate the cells on ice for a short time (to lull them into a false sense of security), and then transfer them to a hot water bath (42°C) for thirty seconds. Thirty seconds is all we need for the bacteria cells to panic and scream "WHAT IS HAPPENING TO ME?" This prompts the bacteria, because they are stressed, to take up any DNA in their environment. Well good thing the only DNA in their environment is the plasmid we gave them! Through the heat shock, a large amount of bacteria should have taken up our plasmid. We then grow the bacteria over night while they recuperate, divide exponentially, and make copies of our plasmid. This process is summarized by the cartoon below:

Apparently I've trained my bacteria really well...

...look what they did last night!

They're super neat! ^,^

What's up PCR reaction that finally worked!?

BAM! LOOK AT THOSE BANDS!

Three PCR reactions, three beautiful bands. Looks like digesting the insert and then amplifying it really did the trick. This means I can use my PCR reaction to transform some bacteria!

The idea is to insert our PCR reaction into a plasmid vector, which we then transform into E. coli. We do this via heat shock, which causes the bacteria cells to take up plasmids. We then will grow the E. coli, conduct a plasmid prep to collect all of the plasmids they grew, and then I will perform another digest to recut out the terminator region (the PCR product). This way, I can be sure the restriction sites were correctly added onto the terminator region, which I need in order to insert it into the rest of the plasmid I already have.

I'm transforming the cells as I write this, and will be back soon to give updates. Yay science!