Success!

This is Erin reporting again after a busy week. Good news! The plates that we thought had no growth just took a little longer. Colonies appeared after two days.

These colonies were patched onto new plates to verify they were actually the XL-gold ultra competent with the pUCP28T plasmid with all five mutations. The appearance of colonies indicates that the PCR site-specific mutations worked and were transformed into the cells provided with the kit. The patched colonies also grew so we can assume the process is highly efficient.

This however might be a problem. There is a potential that kit works too well and all five of the mutations were incorporated into the plasmid and transformed into the ultra competent cells. While that is the object of the kit and great that the efficiency is so high, we are trying to determine which of the five mutations causes the temperature sensitive phenotype. We have decided to take a new approach and test each mutation individually. (Recall the original work can be found here: http://aem.asm.org/cgi/content/full/74/4/1064?view=long&pmid=18156318).  Individual PCRs were run for each of the primers. New mutated plasmid DNA is transformed into the ultra competent cells and plated. All but one of the mutations showed colonies after two days. The mutations that have colonies were patched onto new plates. The patched clones from the transformed cells with all five mutations will be saved and kept at my bench.

The next step is to isolate the plasmid and electroporated into our bacterial strain of interest.

I hope to report back in a week in more detail.

Thank you for reading and happy father’s day,
Erin

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