Lecture Chapter 7 - Protein structure and functionChapter 9 - Gene regulation in prokaryotes(And... The PCR Song)

We finished chapter 7, and the third stop in our road map, on how DNA organization influences protein function. We discussed the most common structural motifs of DNA binding proteins and agents that may denature or even degrade proteins.

We also started chapter 9, on regulation of gene expression in prokaryotes. We discussed the basics of positive and negative regulation and the differences in frequency of gene regulation events at transcription, translation and in between.

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The PCR song

The highlight of the day was, with no doubt, the performance of The PCR Song by students in this class. You can see the video of the original song by clicking here, or accessing the previous entry in this blog.

The exercise, meant to reinforce the reagents and steps of PCR, ended up being delightful and enjoyable one, even leading to the suggestion to use more songs that summarize processes (a molecular biology greatest hits CD?).

I am open tu suggestions and new ideas!

(the mp3 file of the Winter 10-11 Biol 217 Ensemble is available upon request!)

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PCR song

After exam 1, which showed that several people in the class are confused on what is required to perform a PCR and on the steps of the thermal cycle, I decided to reinforce the basics of the procedure that were explained in labs 4 and 5.

Students were allowed to vote on having a quiz on Friday or learning by heart and singing the PCR song. A nearly unanimous decision favoring the latter was reached. The PCR song highlights:

• Who invented PCR
• The main reagents of PCR
• The steps of the thermal cycle
• Some important applications of PCR

Tomorrow (Friday) students will sing the song in class for the opportunity to 1) properly learn the basics of one of the most popular molecular techniques and 2) earn a few points.

The song: Students in previous quarters have found this song useful and some have even used it as a ringtone... (Warning: Cheesy!)

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Module 2Lab 6 - Gel electrophoresis of the pJet1.2+GAPC gene R.E.D.Lab 7 - Sequencing reactions of the GAPC gene

Lab 6

Today we ran the gel electrophoresis of yesterday's R.E.D. to confirm the successful ligation of the GAPC gene from Arabidopsis, which we amplified through nested PCR, with the pJet1.2 plasmid.

The results were used to determine which plasmid DNA purified samples were to be used in lab 7, when setting the sequencing reactions for the GAPC gene.

Lab 7

We added forward and reverse sequencing primers (pJET SEQ F and pJET SEQ R) to the plasmid DNA purified samples that had the GAPC gene insert 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 2-5 weeks, ready to be used in the bioinformatics labs

We will discuss 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 DNA sequencing process.

(Note: Section 2 followed these protocols on Monday)

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Module 2, lab 6 - GAPC gene cloningPlasmid DNA purification ('miniprep')

Today we used the E. coli cultures Salesha and Jess inoculated yesterday to do a small-scale plasmid DNA extraction ('miniprep') using Promega's Wizard® Plus SV Minipreps DNA Purification System. Then restriction enzyme digestions with Bgl II were set to confirm that the insert (GAPC gene) is present in the plasmid (pJet1.2).

(Section 2 did the procedure on Monday [MLK day]. Thanks to Stacy for inoculating the media the day before!)

Here's a recap of what we have done so far in the last couple of exercises:

Lab 5 - Nested PCR of the GAPC gene from Arabidopsis

Lab 6 - Cloning he GAPC gene from Arabidopsis

• Ligation of PCR amplified GAPC gene onto the pJet1.2 plasmid
• Genetic transformation of E. coli with the pJet1.2 plasmid
• Cloning of genetically transformed E. coli
• Minipreps (purification of pJet1.2 plasmid)

Next, running an agarose gel to confirm the results of the R.E.D. (lab 6) and setting DNA sequencing reactions (lab 7)

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Lecture, chapter 7 - Protein structure and function

Today we talked about the quaternary level of structure in proteins, and the nomenclature of proteins that are composed by several subunits.

We discussed a classification of proteins from a molecular biology perspective and also how DNA binding proteins can read information contained in DNA sequences without undoing the double helix.

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