Lab 09 - DNA sequencing

Friday Oct 16 2009

Today we ran gel electrophoreses to confirm if the plasmid extraction (lab 8) was successful and if we had the GAPC gene (from Arabidopsis) insert. In some cases we did.

Using the samples that had the insert we mixed the miniprep DNA with forward and reverse sequencing primers (pJET SEQ F and pJET SEQ R), and put them in a 96-well plate. The plate will be shipped to the DOE Joint Genome Institute (JGI) to be sequenced as part of their Sequencing Training Program (STR). The results should be in in two weeks, ready to be used in the bioinformatics labs

The report from lab 9 will be merged with the report of lab 15 (Bioinformatics)

While the gels were running we discussed the DNA sequencing technique most commonly used: Dye-terminator sequencing, a modification of Sanger's chain termination sequencing protocol, which allowed the automation of the sequencing process.

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Lab 08 - Ligation & Transformation

Today we almost finished lab 08, the ligation and transformation module (ligation of the Arabidopsis GAPC gene into the pJet1.2 plasmid; transformation of E. coli).

We did a plasmid DNA extraction (minipreps) from the bacterial cultures we did last week, and initiated a restriction enzyme digestion with BglII to confirm if we have plasmid DNA and the insert we are interested in. Tomorrow we will run an agarose gel electrophoresis to confirm the results.

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Lecture, chapters 7 and 9 - Protein structure and function & Gene regulation in prokaryotes

Today we finished chapter 7, on protein structure and function. We discussed the main structural motifs found in DNA-binding proteins, and talked about protein denaturation.

We also started covering chapter 9, on gene regulation on prokaryotes, focusing on regulation at the transcription level. We discussed the importance of gene regulation and some of the key players involved in the process.

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Lecture, chapter 7 - Protein structure and function

Today we covered most of chapter 7, on protein structure and function. We further discussed the secondary structure of proteins (α-helices and β-sheets), and we talked about the tertiary and quaternary structures as well.

In terms of function we mainly discussed how DNA-binding proteins can read the information in the double helix without breaking the hydrogen bonds between bases.

Reminder: The first draft of the review paper is due tomorrow at noon. Send me an electronic file (preferably a Word document) before then.

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