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!

Sometimes Digests Possess The Answer

Today I'm running a digest on a plasmid in an attempt to cut out a portion of DNA.

This plasmid is the nitrite reductase (NiR) gene terminator region cloned into a vector (called pCR4). Just outside of where the terminator region should be inserted is a series of primer points and restriction sites.  EcoRI restriction sites flank just outside the cloning site, and as such I'm running an EcoRI restriction digest. The digest only takes an hour at 37°C on a heating block (right), and is a simple reaction containing just restriction enzymes, buffer, DNA, and water.

I took a quarter of the restriction digest reaction (5 of the 20µl), added 1µl loading dye, and ran the reaction on a 2% agarose gel:


Here we can see in the second lane my digest. The really bright band is the vector (most of the DNA in the plasmid) and the smaller band (further along on the gel) in my insert, just under 1,000 base pairs. The ladder (the top and 5th lanes) is a 1kb ladder--moving right to left, the smallest band is 500bp, then 1kb, 1.5kb, 2kb, 3kb, and 4kb, etc. I like to use a 1kb ladder most of the time because it's really easy to use, and I can quickly tell which band is which because the 3kb mark is the brightest in the ladder. in my gel here, the vector sequence is larger than 3kb, and roughly equal to the 4kb band (which was expected). The insert that was digested out in the reaction is just short of 1kb (which was also expected.

I then ran the rest of the reaction on a second gel (which was a sort of a waste, but I always want to check my reaction before I look to do anything else with it), because my digest worked. I wanted to cut out the smaller band and use that in a PCR reaction, to make it easier for the primers to amplify them. Since I've been having trouble getting the primers to work on this plasmid, we decided we might as well try this. So I ran the rest of the digest reaction in lanes 2 and 3--you can see where I cut out the bands, which I did with a razor blade (which is the black silhouette on the right).

I then melted the agarose gel that I cut out with my DNA band, and cleaned up the solution. By binding the DNA to a small filter, I could clean the DNA and remove the gel. I then yielded (what I hope is) the digested plasmid insert. I'll use this cleaned up insert from the gel in a PCR reaction overnight tonight and hopefully get some better results.


Below is a cartoon representing the NiR sequence I'm working with. For now, I'm just focusing on the terminator region, which is the 3' untranslated region (UTR) sequence just after the stop codon. By sequencing the DNA we're working with or looking up the desired DNA sequence online at a databank website, we can model the DNA sequence and figure out where the start and stop codons most likely exist. We can then make primers to amplify specific regions along that DNA.

The blue rectangle represents the NiR terminator that we're amplifying: it's just a little bit longer on either side of the actual 3' UTR, which means we're sure to amplify the entire 3' UTR.