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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Lecture, chapter 7 - Protein structure and function

Today we started the final chapter on the third stop in our road map, specifically on how genetic information affects protein function.

We discussed the structure of amino acids and how they are linked into polypeptides thanks to peptide bonds (a special case of covalent bonds).

We also discussed the basics of the four levels of organization in proteins, but focused specially on the secondary and tertiary levels of structure. We discussed the importance of hydrogen bonds in maintaining alpha-helices and beta-sheets, an how these are folded into 3-D structures that are crucial for protein function.

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Module 2, lab 6 (section 1) GAPC gene cloning

Click here for info on the same procedure, performed by students in section 2.

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Module 3, lab 9 (section 1) Hydrophobic Interaction Chromatography (HIC) of GFP

Click here for info on the procedure performed by students in section 2.

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Lecture, chapter 6 - Transcription

DNA looping and interaction of general and specific transcription
factors with RNA polymerase in eukaryotic cells during transcription
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Today we finished chapter 6, on transcription.

We discussed how the RNA polymerase recognizes specific DNA sequences in the promoter of prokaryotic DNA, and how it forms a transcription bubble in order to read the template strand on the DNA and synthesize the RNA molecule. We commented how the terminator causes the formation of a hairpin in the RNA transcript that causes the RNA ploymerase to "fall off" the DNA.

We highlighted the differences in transcription between prokaryotes and eukaryotes:

• the use of three kinds of RNA polymerase (I, II, and III) for transcribing different kinds of genes
• the use of general and specific transcription factors
• the role of enhancer regions in aiding the transcription process
• the need for DNA looping and the use of a mediator complex for all the transcription factors to interact with the transcription apparatus

CLICK HERE to watch a good Youtube video on transcription, or HERE for a video from the Dolan DNA Learning Center!

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Module 3, lab 9 (section 2) Hydrophobic Interaction Chromatography (HIC) of GFP

Yesterday we performed a Hydrophobic Interaction Chromatography (HIC) to separate the green fluorescent protein (GFP), produced in our bacterial cultures, from other proteins commonly found in bacteria.

A sample of bacterial culture was concentrated and then resuspended in a solution in which they were lysed. The high salt solution, containing all the proteins found in the bacteria, was then passed through a hydrophobic interaction column where molecules of GFP bound to the hydrophobic beads. The high salt solution increased the hydrophobicity of GFP by further exposing its hydrophobic amino acid residues.

A series of washes with buffers of decreasing salinity allows proteins with various levels of hydrophobicity to gradually unbind from the beads and be collected in a test tube. By switching collection tubes each time a buffer is added, different proteins can be collected. One of them was GFP and the tube in which it was collected glowed with a green color (of course).

Diagram of Hydrophobic Interaction Chromatography (HIC)

GFP molecules are represented by black triangles

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Module 2, lab 6 (section 2)GAPC gene cloning

Yesterday we started the process of cloning the GAPC gene from Arabidopsis, which we amplified via nested PCR in lab 5. The lab was divided in three main steps

• Ligation (of GAPC gene on to the pJet1.2 plasmid)
• Preparation of competent cells
• Genetic transformation of E. coli

We spent most of the lab manipulating bacteria to make them competent (i.e. get them ready to uptake extracellular naked DNA). Once this was achieved, the GAPC gene from Arabidopsis, obtained via nested PCR, was ligated to the pJet1.2 plasmid.
The plasmid was then used to genetically transform E. coli, which were spread on LB agar/Amp/IPTG plates and incubated.

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Exam 1

Exam stats:

(click on pic for full size image)

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Module 3, lab 08 (section 1)Genetic transformation of E. coli with the pGLO plasmid

We followed the same process we followed with section 2.

For more information click here.

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Module 2, lab 5 (section 1)GAPC gene nested PCRGel electrophoresis (PCR round 2) and PCR purification

Today we ran the agarose gel electrophoresis for the second round of the nested PCR of the Arabiodopsis GAPC gene (which we will clone, and eventually sequence), and also purified the reaction products, in order to eliminate leftover reagents and keep only the DNA.

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Lecture, chapter 6 - Transcription

Today we started chapter 6, on transcription, covering the basics of the process.

We discussed how to decide which are the template (sense) and coding (anti-sense) strands in DNA, the reasons for which some genes are expressed all the time (housekeeping genes) and some others are expressed only some times, and included some transcription-specific terminology.

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Module 3, Lab 08 (section 2)Genetic transformation of bacteria with the pGLO plasmid

Aequorea victoria, original source of the green fluorescent protein (GFP)

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Today we used the pGLO plasmid to genetically transform Escherichia coli.

pGLO is a plasmid that has been engineered to contain the Green Fluorescent Protein (GFP) gene, originally isolated from the jelly Aequorea victoria. GFP produces a green fluorescence when excited by blue or UV light.

In order to make the GFP gene a functional one it has been engineered so the sugar arabinose triggers the production of the protein. The genes in the arabinose operon (araB, araA, and araD) have been replaced by the GFP gene. Such genes encode proteins that break down arabinose when it is present in the environment, so they are expressed only if this is the case. The regulatory sequence has been left intact, so in the engineered operon the presence of arabinose turns on the GFP gene and, therefore, GFP is produced.

Another feature of the pGLO plasmid is the presence of the beta-lactamase gene, which provides resistance against the antibiotic ampicillin.

The bacteria were transformed through the heat shock technique, and then plated on LB agar plates containing:

• Just LB (lysogeny broth)
• LB and ampicillin
• LB, ampicillin and arabinose

Plates are being incubated for 24 hours at 37ºC.

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Lecture, chapter 5 - DNA replication

We finished the chapter on DNA replication.

We discussed the process in which the DNA polymerase complex actually replicates DNA, including the DNA looping that allows both strands to be synthesized at the same time.

We also covered eukaryotic DNA replication, emphasizing the differences with prokaryotes.

Watch the following video or access this link to understand the main features of the replication process

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Section 1Module 2, Lab 05 - GAPDH nested PCRAmplifying the GAPC gene

Same lab as with section 2. Click here for information on it.

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Lecture, chapter 5 - DNA replicaiton

Today we started chapter 5, on DNA replication, and the third stop in our roadmap, on how genetic information is preserved and inherited.

We started a discussion on how DNA is replicated in prokaryotes, including the concepts of replication fork and replisome. We described the role of several enzymes on the process of DNA replication (DNA gyrase, DNA helicase, single strand binding (SSB) proteins, primase, DNA polymerase), and the fact that there are a leading and a lagging strand during the replication process, the latter being extended in bursts forming Okazaki fragments.

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Section 2Module 2, Lab 05 - GAPDH nested PCRAmplifying the GAPC gene

Thale cress, Arabidopsis thaliana
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Today we started the exercise in which students will learn the basics of nested PCR. We will work with the gene that encodes one of the GAPDH isomers, GAPC, in the thale cress (Arabidopsis thaliana), the model organism of plants. Some people call it "the fruit-fly of plants".

GAPDH is an enzyme in charge of catalyzing one of the reactions in glycolysis. There are several nuclear genes that encode GAPDH isomers (proteins with different amino acid sequences but with the same function), and we are targeting the gene GAPC in the A. thaliana genome. We ran a first round of PCR, with our initial primers, and on Wednesday we will do the second run, with the nested primers.

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Section 2Module 1, lab 4 - PCR of the PV92 Alu insertion locus

Today section 2 completed lab 4 in module 1. For more information click on PCR of the PV92 Alu insertion locus

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