Sup July? SUP SUCCESSFUL TRANSFORMATIONS?

I've been having trouble transforming E. coli cells with my plasmid vector, within which is a small DNA fragment (my NiR terminator) that I will want to restriction cut out. By transforming bacteria with the plasmid vector, I'll make additional copies of the plasmid and be able to freeze and save the plasmid for later use if necessary.

My lab mates and I spent several weeks trying to figure out why our transformations were doing so poorly and why we were receiving such low plasmid yields from transformed bacteria. I myself figured out that one problem was the ampicillin used to make the agar plates upon which we grow our bacteria had degraded over time, and that the antibiotic was not selecting strongly enough to weed out bacteria with plasmids and bacteria without plasmids. This is the reason why we were not getting good plasmid yields and another reason why our bacteria were not growing when transferred from "old" plates to new agar plates with freshly made ampicillin.

We also concluded that the bacteria cells we were transforming were not up to par to yield the results we needed, so we ordered some new transformation kits.

But in order to successfully clone PCR product into a plasmid vector to transform into bacteria, the PCR product needs to be freshly made. In order to get new PCR product, I re-amplified older PCR product in the same reaction I ran before. I ran four different reactions using the PCR DNA in four different DNA concentrations: 1:1, 1:10, 1:100, & 1:1,000 (lanes 2, 3, 4 & 5 in the picture below respectively). This way I can determine which reaction had too much starting DNA and too little. After my reaction, I ran part of it on a gel to see how each reaction went. I definitely got much larger yields in the 1:1 & 1:10 dilutions (there was probably too much DNA even), so I used the second dilution (1:100, lane 4) to clone into the plasmid vector.

I used PCR product from lane 4 to clone into a vector plasmid for the transformation.

Using the vector plasmid, I transformed them into the bacteria and let them grow over night on an agar plate. I then performed a colony screen, which is a PCR reaction using single bacteria colonies to supply the DNA. That PCR reaction yielded the below gel:

While this is a slightly messy colony screen gel, several of these colonies should suffice!

What we're seeing in this gel is the molecular ladder at the top and then 10 different colony screen reactions. They're pretty streaky, which is probably because there was a lot of bacterial DNA in each PCR reaction. What I wanted was a single band at around 700 basepairs, which is roughly half way between the two second most right bands on the ladder. As such, lanes 4, 6, 7 & 8 are good candidates for colonies that have my plasmid with the correct insert.

BRB time for the holiday weekend!

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.