Labs 06, 07, and 08

Thursday October 08 2009

Lab 06 - pGLO bacterial transformation

Students analyzed the results of the bacterial cultures in LB, LB/Ampicillin, and LB/Ampicillin/Arabinose agar plates. A colony in the latter will be used to start lab 07.

Lab 07 - Protein purification by chromatography

Students used a colony from the LB/Ampicillin/Arabinose agar plates generated in lab 06 to inoculate an LB/Ampicillin/Arabinose broth tube. The broth was incubated at 37ºC for approx. 24 hours in a shaking water bath at approx. 200 rpm.

Lab 08 - Ligation and genetic transformation

Students prepared competent cells (E. coli) and transformed them with the pJet1.2 plasmid that has the GAPC gene (from Arabidopsis) insert.

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Lecture, chapter 7 - Proteins

We finished chapter 6, on transcription of genes, and went ahead and started chapter 7, on protein structure and function.

We talked about amino acids, the monomers that make up polypeptides, and how they are organized in four levels of structure. We also made the distinction between a polypeptide and a protein. We took a closer look to the primary and secondary structures of polypeptides (and proteins).

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Lecture, chapter 6 - Transcription of genes

Today we covered most of chapter 6, on transcription of genes.

We compared some details of transcription in prokaryotes and eukaryotes, including the kind of RNA polymerases involved in each one, the transcription factors involved in eukaryotic transcription, and some of the components of regulatory DNA.

Reminder: Next Wednesday (Oct 14) is the due date for the first draft of the review paper. The more advanced the paper the more feedback I'll be able to give you!

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Lab 06 - Bacterial genetic transformation with pGLO Lab 08 - Ligation and transformation (gene GAPC)

We performed the genetic transformation of E. coli cultures with BioRad's pGLO™ plasmid, engineered to contain the green fluorescent protein (GFP) gene , originally isolated from the crystal jelly Aequorea victoria.

We also performed the ligation of our GAPC gene (obtained in lab 05) with BioRad's pJet1.2 plasmid. In the next lab we will genetically transform bacteria, just as we did earlier today.

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Lab 05 - GAPDH Nested PCR

Thursday, October 1st 2009

We ran the gels for the nested PCRs we performed last week.

Due to failure of starter bacterial cultures we couldn't start lab 6, on bacterial genetic transformation, but we did talk about the main topics the lab is related to: Gene regulation, antibiotic resistance, and genetic transformation.

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Exam 1

Today we had our first partial exam.

Results and stats:

(click image for full size view)

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Lecture, Chapter 6 - Transcription

After finishing chapter 5, on the few details that differ in eukaryotic DNA replication compared to that in prokaryotes, we started chapter 6, on transcription.

We talked about the basics of the synthesis of transcripts, and got acquainted with some new terms like cistron, open reading frame (ORF), monocistronic and polycistronic mRNAs, and operon.

Tomorrow: Exam 1...!!!

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Lab 05 - GAPDH Nested PCR

Arabidopsis thaliana

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Today we started the exercise in which students will learn the basics of a nested PCR. We will work with the gene that encodes one of the GAPDH isomers, GAPC, in 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 tomorrow, Friday, we will run the second run, with the nested primers.

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Lecture, Chapter 5 - DNA replication

We continued covering chapter 5, on DNA replication. We finished talking about DNA replication in prokaryotes, topic in which students should have an understanding of the replication fork, including the functioning of all the enzymes involved in the process.

We started talking about DNA replication in eukaryotes, organisms in which some differences are found, mainly because of the linearity of chromosomes. In prokaryotes chromosomes are circular.

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Lecture, Chapters 4 and 5 - Genes, Genomes, and DNA & DNA Replication

Today we covered the end of chapter 4, focusing mainly on the mechanisms prokaryotes and eukaryotes use to supercoil their DNA.

Then we started with chapter 5, on DNA replication. We introduced the concept of replication fork and went over some of the issues the cell has to solve in order to get supercoiled DNA to replicate.

Reminder: We are in week 3 and students should be meeting with me to decide the topic of the review paper.

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Lab 04 - Detection of genetic modification in crops

Friday Sep 18 2009

We started the exercise in which we will test corn and soy samples students brought to the lab to see if they have been genetically modified (if they are Genetically Modified Organisms or GMOs).

We extracted DNA from corn and soy leaves, as well as from a certified non-GMO seed provided by Bio-Rad with the kit. We set up PCRs using primers that will amplify de 35S promoter of the cauliflower mosaic virus (CaMV 35S) and the nopaline synthase (NOS) terminator of Agrobacterium tumefaciens, which are present in about 85% of all modified crops in the U.S. As a positive control for the presence of DNA, we also used primers that amplify the photosystem II chloroplast gene, which should be present in all plants, regardless of genetic modification.

Next week we will run an agarose gel electrophoresis to confirm the results.

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Lab 03 - PV92 PCR (postponed)

Thursday Sep 17 2009

Due to the power outage in Ada and subsequent evacuation of Meyer Hall and the Mathile Center, the lab has been postponed until weeks 09 and 10. That way we will be able to still do the lab without disrupting the flow of the following labs, which sequence is of greater importance than that of the PV92 PCR lab.

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Lecture, Chapter 4 - Genes, genomes, and DNA

Today we finished covering chapter 3, on nucleic acids and proteins, and started covering chapter 4, on genes, genomes, and DNA (2nd stop on the 'roadmap': How DNA is organized in organisms, and how such organization affect its function).

We also had our first quiz, and I announced that tomorrow we are having another one, so we can keep with the average of 1 quiz/week.

Reminder: List of topics for the review paper is due next week. Each team (of 2 people) should make an appointment with me to go over the list, and pick the topic.

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Labs 01 and 02

Thursday Sep 10

Using the micropipette

On Thursday students got acquainted with their new best friend, the micropipette. We did a couple of exercises to make sure measurements were made accurately and that students learned how to use a combination of micropipettes to measure different values. The report was handed in at the end of the lab.

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Friday Sep 11

Restriction Enzyme Digestion (RED) and analysis of lambda DNA through gel electrophoresis

In this lab we used three different restriction enzymes EcoRI, PstI, and HindIII to digest (cleave) DNA from the lambda bacteriophage. As a DNA marker, or DNA "ladder", we used a sample of lambda DNA pre-digested with HindIII.

Students will measure the distance bands in the gel migrated and will infer the size of the different bands based on such information.

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Lecture, Chapter 3 - DNA, RNA, and Protein

Today we had our first lecture, and we covered most of chapter 3, on the structure and basic function of DNA, RNA, and proteins.

We covered the basic structure of nucleic acids and some of the main differences between DNA and RNA. We also mentioned a few key characteristics of the structure of chromosomes.

Next week we'll finish chapter 3 and start chapter 4, on genes, genomes and DNA ("how DNA is organized in organisms" according to the road map [see Power Point presentation from first meeting]).

Reminders:

• Two quizzes next week
• Topics of choice for review paper in two weeks

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Fall 2009...!!!

Welcome to the Fall 2009 version of Introduction to Molecular Biology...!!!

In today's session we went over the syllabus and explained some of the main components of the class. Besides the syllabus hard copy you have by now you can download it from the class WebCT site as well as from the p-drive (under 'a-cordoba').

Reminders:

• You must read chapters 1 and 2 in the textbook, or be comfortable with the material. We will not lecture on those chapters but the information is important as background for the rest of the course.
• Pair up with a classmate in order to prepare for writing the review paper in a topic on molecular biology. As of now there are 22 registered students in the class, so there is no need for a team of three. Choose wisely, since part of your grade will depend on your team-mate (see syllabus).
• Prepare a list of three topics you would like to develop in the review paper and the reasons for choosing them. By week 3 you must meet with me, out of class, so we can choose one of the topics, based on your reasons, relevance for your future career, and relevance for the class.

Any ideas to improve this blog will be greatly appreciated. What would you like to see posted here?

Have a great quarter!

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Links for Lab 15 (Bioinformatics)

• National Center for Biotechnology Information (NCBI)
• DNA Data Bank of Japan (DDBJ)
  
• European Bioinformatics Institute (EBI)

• GenBank statistics
• Clustal W
• Phylogeny software
  

• NCBI's Coffee Break

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Lectures - DNA repair + Control of gene expression

Between last monday and today we have finished the section on DNA repair and have covered most of the section on control of gene expression.

Last Monday (Jan 26) we closed the DNA repair section, covering the concept of emergency DNA repair: When extensive damage in the DNA is detected by RNA polymerase and is repaired not just by the regular DNA polymerase, but also by a battery of DNA polymerases that are less accurate, but more specific for a type of damage. They also lack proofrweading capacity, so the likelihood that there will be mistakes during the repair process is higher than with other DNA repair mechanisms.

Between Friday (Jan 30) and today, we have been studying the basics of control of gene expression. So far we have focused on transcriptional control, and on Monday we will focus on mechanisms of post-transcriptional control.

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Lab 12 - Small-scale plasmid DNA purification (minipreps)

Thursday Jan 29 2009

We went back to the bacterial cultures we had of transformed bacteria (E. coli), to reverse (in a way) the process we started. In this case we want to isolate the plasmid (Bio-Rad's pGLO) that we used to genetically transform the bacteria. In the process of cloning DNA this is one of the steps you follow to study the DNA segement of interest, in our case the GFP gene contained in the pGLO plasmid. We made zillions of copies of it, and now we have to extract it from the bacteria to analyze it.

We used Promega's Wizard® Plus SV minipreps DNA purification system. An easy to use kit to purify plasmid DNA in a lab like the one we have available.

Next week we'll run a confirmation gel and perfomr a restriction enzyme digestion (RED) of the pGLO plasmid.
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Lab 11 - Polyacrylamide Gel Electrophoresis (PAGE) of GFP samples

Thursday Jan 29 2009

This lab should have taken place on Wednesday Jan 28, but classes were cancelled due to weather.

Polyacrylamide Gel Electrophoresis (PAGE) is a technique used for separating polynucleotides or polypeptides that are very similar in size, providing greater resolution than with an agarose gel.

We used PAGE to measure the size of a protein, GFP. If we follow the series of "make believe" in which we are dealing with a new protein, this would be a step to find more information about it. Protein size is quantified in Daltons (Da), a measure of molecular mass. One Dalton is defined as the mass of a hydrogen atom, which is 1.66 x 10-24grams (g).

PAGE is used for separating proteins ranging in size from 5 to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel. Agarose gels can also be used to separate proteins, but they do not have a uniform pore size, so they are optimal only for electrophoresis of proteins that are larger than 200 kDa. We ran two types of PAGEs: Native and denaturing (a.k.a. SDS)

Proteins can have varying charges and complex shapes, therefore they may not migrate into the gel at similar rates, or at all. In native gel electrophoresis the proteins being separated differ in molecular mass and intrinsic charge and experience different electrophoretic forces dependent on the ratio of the two. Because of different charges and tertiary structure proteins of the same mass may migrate at different rates.

In SDS gel electrophoresis proteins are denatured (linearized) in the presence of a detergent such as Sodium Dodecyl Sulfate (SDS) that coats the proteins with a negative charge. The resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like long rods instead of having a complex tertiary shape, the rate at which they migrate in the gel is relative only to its size (molecular weight) and not its charge or shape.

We will be able to compare teh results in both gels, and if GFP has any activity in either one of them (through pictures taken under UV light).
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