After several weeks of DNA extractions, I finally moved on to the next steps in my thesis research!!
The end goal of my thesis project is to create a detailed profile of the bacterial communities in Lake Matoaka based on bacterial gene sequences. I began with a giant mix of environmental DNA from almost a year’s worth of sample collections. The next step is to get that DNA into a specific, sequenceable form and get a lot of it. Using PCR amplification of the 16s rRNA gene (a gene that is highly conserved in all bacterial species), I can get a mix of specific and sequenceable amplicons. However, I only have the ability and means to sequence a few genotypes out of the vast array of 16s amplicons. How can I narrow down the field, if you will? The solution: TOPO-TA Cloning Kits for Sequencing (aka my new bffs). The TOPO-TA Kit allows me to perform ligation and transformation reactions to narrow the vast amount of DNA to just a few purified sequences. The kit works in two distinct steps.
The first step is the ligation step. A mixture of the 16s amplicons (in my case from the samples collected in June and December of 2009) is added to a vial containing plasmid vectors. These vectors are prepared in such a way as to allow for insertion of the 16s amplicons at A to T overhangs into the plasmid. Imagine you are building a circular toy train track. You’ve almost completed your circle and are missing one piece. Each piece fits together by overhanging pieces. Click the missing train piece into place and you’ve created a perfect, circular train track. In this case, the plasmid is the almost completed train track and the 16s amplicon (insert) is the missing piece.
Once ligation is completed, transformation is next. Chemically competent E. coli cells – cells that have been treated so as to make their membranes more permeable – are now mixed with the insert-containing plasmids. A combination of incubations on ice and heat shocks is used to force the competent bacteria to take up the plasmid vectors. The transformed E. coli are then spread onto selective media plates and incubated overnight.
Did the transformation work? How do I get those inserts back out of the E. coli? How do I sequence the inserts? These questions and more will be answered in my upcoming posts! Stay tuned!