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!