USA!

As they say in Afrikaans: Hallo. Erin here with a brief plug:

I am going to start this entry by congratulating team USA in the FIFA world cup. The matches have been unbelievable and for anyone who hasn’t seen the last two games USA had a disallowed goal in each game and still managed to progress to the second round of 16 in which they play this morning (fingers crossed their streak will continue).

Enough about soccer, the lab has also been buzzing like the vuvuselas of the world cup. If you can recall last week I used Stratagene’s QuikChange Multi Site-Directed Mutagenesis Kit to insert different point mutations in the wild type pUCP28T plasmid. Four out of the five transformants with individual point mutations did grow on selective media. Clones were patched for verification. (Patched: single colonies are transferred from the original plate to a second plate overlaying a grid. The purpose is to isolate single clones and be able to have them numerically ordered for later use.)

All the patched clones also grow up on selective media. At this point the plan has taken on a new face. Instead of jumping straight to trials in our bacterium of choice, the plasmid DNA will be sent away for sequencing to verify the change in DNA sequence and to ensure no other random mutations. Twenty-two sequence requests were sent in, thankfully twenty-two requests were returned with good results. After analyzing the sequences we have a slight snafu. The “control plasmid” (the plasmid with the five mutations and temperature sensitive phenotype) resembled the sequence for the wild type plasmid. Other than this, all the other sequences were as predicted with no random mutations.

From here we try to isolate the correct control plasmid (a different strain may contain the Ts plasmid) and the last point mutation incorporated into the wild type background. After these sequencing results it’s on to trials in Burkholderia.

It is all a matter of pushing forward along with a little trial and error.

GO USA!

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