Today we lysed our bacteria open, extracted all the molecules inside them, including all their proteins, and then purified our protein of interest, the GFP.
We performed a protein chromatography using Bio-Rad's disposable columns. Our task was separating the GFP from the thousands of other proteins that we would find in a bacterium. The resin used in these columns were chosen to separate specifically the GFP by Hydrophobic Interaction Chromatography (HIC).
In HIC the resin is very hydrophobic. The salty buffer used causes proteins to change their 3-D conformation exposing the hydrophobic amino acids, wich stick to the hydrophobic resin. The GFP sticks especially strongly to the resin. Then we did washes with solutions of decreasing salinity so other proteins that happened to be less hydrophobic than the GFP recovered their original conformations, un-bound from the resin and were washed out. At the end we used a low salinity solution that allowed thr GFP to recover its original 3-D conformation making its hydrophobic amino acids un-available and separating from the resin.
Following this protocol we purified the GFP exclusively, and that's why our little tubes glowed when exposed to UV light.
-------------------------
Friday, October 10, 2008
Thursday, October 9, 2008
Miscellaneous news and notes
Somebody asked me about the questions for lab 4/5. THat lab was due last week, but here's a summary of the questions that I want for the last few labs:
Lab 4/5: All five questions on page 56 of that lab maual
Lab 6: Questions 1, 4, and 5 on pages 43 and 44 of that manual. Questions on page 54 don't have to be answered in the questions section, but if you can answer them for your self you an write a solid discussion. Use those questions as suggestions to how to write the discussion.
Lab 7/8: Questions on page 38 of that manual. Some people answered more than those, and that will not hurt. It will only help!
Lab 9/10:
Band sizes in the ladder of the GMO lab (lab 6)
Lab 4/5: All five questions on page 56 of that lab maual
Lab 6: Questions 1, 4, and 5 on pages 43 and 44 of that manual. Questions on page 54 don't have to be answered in the questions section, but if you can answer them for your self you an write a solid discussion. Use those questions as suggestions to how to write the discussion.
Lab 7/8: Questions on page 38 of that manual. Some people answered more than those, and that will not hurt. It will only help!
Lab 9/10:
- In your own words, describe cloning
- What is a bacterial colony?
- In your won words describe hydrophobic interaction chromatography (HIC) and identify its purpose in this lab
Band sizes in the ladder of the GMO lab (lab 6)
- 1000 bp
- 700 bp
- 500 bp
- 200 bp
- 100 bp
LAB - GFP purification through chromatography
Today we started the process to purify the Green Fluorescent Protein (GFP) from the bacteria we transfromed last week.
The process was really short: Just picking a colony from each of the LB/amp/ara and LB/amp plates, usit to inoculate a tube with a few mLs of LB/amp/ara broth, and sticking the tubes in a shaker/incubator at 32ºC and an undisclosed number of RPMs. Undisclosed, because the knob is not in RPMs, but through experience I would estimate that we set it at about 200 rmps.
The tubes were set so they are in a diagonal position, so the surface area of the medium is larger than in vertical position, promoting a better uptake of O2 from the air, thus enhancing the efficiency of bacterial growth.
Tomorrow morning we will do the actual chromatography to isolate the GFP.
-----------------------------------
The process was really short: Just picking a colony from each of the LB/amp/ara and LB/amp plates, usit to inoculate a tube with a few mLs of LB/amp/ara broth, and sticking the tubes in a shaker/incubator at 32ºC and an undisclosed number of RPMs. Undisclosed, because the knob is not in RPMs, but through experience I would estimate that we set it at about 200 rmps.
The tubes were set so they are in a diagonal position, so the surface area of the medium is larger than in vertical position, promoting a better uptake of O2 from the air, thus enhancing the efficiency of bacterial growth.
Tomorrow morning we will do the actual chromatography to isolate the GFP.
-----------------------------------
Tuesday, October 7, 2008
Chromosomes
Yesterday and today we covered pages 202-219 and 233-245 in chapter 4. We talked mostly about chromosomes, how they are composed, what are their main features, how they are studied and how they are used to study the genomes of some organisms.
Reading for next week: pages 245-260
Quiz # 6 Q&As:
1. What are the three main components of a nucleotide?
Nitrogenous base, phosphate group, sugar
2. Why are the ends of a DNA strand called 5’ and 3’?
Carbons 5' and 3' in the sugar of a nucleotide are the ones that form covalent bonds with other nucleotides. At the ends of a DNA strand a 5' carbon (more exactly the phosphate group associated to it) and a 3' carbon will be open, and potentially ready to bind to another nucleotide.
3. What does it mean that DNA strands are antiparallel?
The 5' end of a strand matches up with the 3' en of the other
4. What does it mean that DNA strands are complementary?
Because the complementarity of the nitrogenous bases once you have the sequence of one strand of DNA you can infer the sequence of the complement.
The association between DNA and some proteins (mainly histones). They very basis of chromosomal structure and function.
---------------------
Quiz # 7 Q&As:
1. What are homologous chromosomes?
In diploid organisms they are pairs of chromosomes that have the same genes. One of the chromosomes is inherited from the father, the other from the mother.
2. What is a karyotype?
The organized set of chromosomes of a given organism. They are stained and homologous chromosomes paired so they can be studied.
3. What is a transposon?
A fragment of DNA that changes locations in a genome, or that it replicates it self so the copy can insert itself somewhere else in the genome.
4. Mention three kinds of transposons and explain one
LINEs, SINEs, repetitive sequences (miscrosatellites, minisatellites), retroviral - like sequences (explain either one of them)
5. What is the name of the histone complexes around which DNA wraps in the chromosomes?
Nucleosomes
----------------------------
Reading for next week: pages 245-260
Quiz # 6 Q&As:
1. What are the three main components of a nucleotide?
Nitrogenous base, phosphate group, sugar
2. Why are the ends of a DNA strand called 5’ and 3’?
Carbons 5' and 3' in the sugar of a nucleotide are the ones that form covalent bonds with other nucleotides. At the ends of a DNA strand a 5' carbon (more exactly the phosphate group associated to it) and a 3' carbon will be open, and potentially ready to bind to another nucleotide.
3. What does it mean that DNA strands are antiparallel?
The 5' end of a strand matches up with the 3' en of the other
4. What does it mean that DNA strands are complementary?
Because the complementarity of the nitrogenous bases once you have the sequence of one strand of DNA you can infer the sequence of the complement.
The association between DNA and some proteins (mainly histones). They very basis of chromosomal structure and function.
---------------------
Quiz # 7 Q&As:
1. What are homologous chromosomes?
In diploid organisms they are pairs of chromosomes that have the same genes. One of the chromosomes is inherited from the father, the other from the mother.
2. What is a karyotype?
The organized set of chromosomes of a given organism. They are stained and homologous chromosomes paired so they can be studied.
3. What is a transposon?
A fragment of DNA that changes locations in a genome, or that it replicates it self so the copy can insert itself somewhere else in the genome.
4. Mention three kinds of transposons and explain one
LINEs, SINEs, repetitive sequences (miscrosatellites, minisatellites), retroviral - like sequences (explain either one of them)
5. What is the name of the histone complexes around which DNA wraps in the chromosomes?
Nucleosomes
----------------------------
Friday, October 3, 2008
LAB - Genetic transformation - making E. coli glow and survive ampicilin
Genetic transformation is the process through which a cell takes up, and expresses, a new piece of genetic material. Usually the organism (e.g. a bacterium) is provided with a new trait that is identifiable after transformation.
In this lab we transformed a strain of E. coli with the plasmid (a piece of circular DNA) pGLO containing the genes Beta Lactamase (ampicilin [an antibiotic] resistance) and Green Fluorescent Protein (GFP), a protein naturally found in the bioluminescent medusa ("jellyfish") Aequorea victoria.
The plasmid is forced into the bacteria through heat shock (42º C - 50 sec). The bacteria will take the plasmid, which contains several genes, including the ones of interest, and it will express them. In the presence of the sugar arabinose (in the culture medium) the GFP will be turned on and expressed, causing the bacteria to glow under UV light.
A couple of useful pics:
--------------------------------
Tuesday, September 30, 2008
Structure and function of nucleic acids
Today we covered pages 195-201 of the book. The chapter starts with DNA structure and function, although we threw in a little bit of RNA structure and function as well. The videos about transcrption and translation didn't work in the presentation I had in class, but I'll post them in this blog shortly.
Next reading: Chromosomes - pages 202-219 and 233-245
Quiz #5 Q&As
1. What are the three main steps of PCR?
Denaturation, annealing, extension (a.k.a. elongation)
2. What are the building blocks of DNA?
Sugar, phosphate group, nitrogenous base
3. What is transcription?
Trnasfer of information from DNA to RNA
4. What is gene duplication?
The copying of a gene and accidental retention of the copy within the genome. The two copies are functional, but one of them may mutate and become a pseudogene and/or eventually become a new functional gene.
5. What is a gene family?
A group of genes that have been generated throufh several gene duplication events, and therefore are similar to eac other in sequence and probably in function.
------------------------
Next reading: Chromosomes - pages 202-219 and 233-245
Quiz #5 Q&As
1. What are the three main steps of PCR?
Denaturation, annealing, extension (a.k.a. elongation)
2. What are the building blocks of DNA?
Sugar, phosphate group, nitrogenous base
3. What is transcription?
Trnasfer of information from DNA to RNA
4. What is gene duplication?
The copying of a gene and accidental retention of the copy within the genome. The two copies are functional, but one of them may mutate and become a pseudogene and/or eventually become a new functional gene.
5. What is a gene family?
A group of genes that have been generated throufh several gene duplication events, and therefore are similar to eac other in sequence and probably in function.
------------------------
The protein synthesis hippie dance
Internet is a great thing and it has interesting sites like Youtube... you can find ANY thing in Youtube. Inspired by the 'PCR song' video, another one of your classmates found this...
What did people do before computer animation existed? They danced. How did people illustrate in a [allegedly] fun way protein synthesis? Well... they got a guy more boring than Professor Frink (from The Simpsons) to explain protein synthesis with a blackboard in 3 minutes and then introduce 'the protein synthesis dance' as performed by a bunch of early 70's hippies (who seemed pulled out from the set of Jesus Christ Superstar) while moving to Iron Butterfly-like music (ask your parents or google it). The image is not that clear, but it sure is amusing...!
----------------
What did people do before computer animation existed? They danced. How did people illustrate in a [allegedly] fun way protein synthesis? Well... they got a guy more boring than Professor Frink (from The Simpsons) to explain protein synthesis with a blackboard in 3 minutes and then introduce 'the protein synthesis dance' as performed by a bunch of early 70's hippies (who seemed pulled out from the set of Jesus Christ Superstar) while moving to Iron Butterfly-like music (ask your parents or google it). The image is not that clear, but it sure is amusing...!
----------------
Monday, September 29, 2008
Literature review paper topics
I have met with most of you and assigned topics for the literature review papers. You are allowed to change topics at any point in time before submitting the first draft (Oct 20th). So far here are the teams and topics:
- DNA fringerprinting disaster victims (Anessa and Chloé)
- Pre-natal genetic manipulation (Amy and Ryan)
- Recombinant DNA technologies in forensic sciences (Jonathan and Katie E.)
- Edible vaccines (Eric and Josh H.)
- Custom drugs (Amanda and Jackie T.)
- Gene therapy (Kyle and Courtney)
- Plant GMOs (Matt and Stuart)
- Evolution of MRSA (CJ and Katie S.)
- Plant disease resistance (Jackie C. and Josh J.)
- The use of DNA in finding missing people (David and Mike)
- The use of DNA in releasing innocent prisoners (John and Brandon)
- Effects and detection of DiGeorge Syndrome (22q11.2 deletion syndrome)(Krystal and Phil)
- Genetic predisposition to disease
- The role of molecular biology in biofuels research
- Pre-natal genetic screening
- Drug resistance in crops
- Disease diagnosis with molecular tools
Contribution from a class mate - The PCR song
I knew about this but I had forgotten about it. One of your class mates found it and wanted to share it with you, so here it goes...
They are nerds, and they obviously grew up in the 80's... oh, and it's a Bio-Rad ad, in case you haven't have enough of them with our lab kits... The PCR song.
The lyrics are actually a good simple review of PCR
------------------
They are nerds, and they obviously grew up in the 80's... oh, and it's a Bio-Rad ad, in case you haven't have enough of them with our lab kits... The PCR song.
The lyrics are actually a good simple review of PCR
------------------
Genomes and diversity - conclusion
Today we finished the introductory topic of the class, in which we talked about mechanisms of generation of new genes, with some emphasis on gene duplication and horizontal gene transfer.
After class there were some doubts about paralogy and orthology, so make sure you understand the difference, or voice your questions in class tomorrow. The difference is simple, but its understanding very important. In case I didn't make my self clear, here's the one line version of the difference: Ortholog genes were separated by a speciation event. Paralog genes were separated by a gene duplication event. It is possible that there is a gene duplication event, and then a speciation event. The two new species will inherit BOTH copies of the gene. Copies 1 and 1 will be orthologous, as well as copies 2 and 2. But copies 1 and 2 will be paralogous within AND among species. Any questions? Please ask in class. No reason to be shy! (It will wake you up. If sleepy BRING COFFEE!)
Reading for Tuesday Sep 30th: Still chapter 4, pages 195-201.
Quiz #4 Q&As
1. Write 4 questions for the next quiz.
Answers will vary
-------------------
After class there were some doubts about paralogy and orthology, so make sure you understand the difference, or voice your questions in class tomorrow. The difference is simple, but its understanding very important. In case I didn't make my self clear, here's the one line version of the difference: Ortholog genes were separated by a speciation event. Paralog genes were separated by a gene duplication event. It is possible that there is a gene duplication event, and then a speciation event. The two new species will inherit BOTH copies of the gene. Copies 1 and 1 will be orthologous, as well as copies 2 and 2. But copies 1 and 2 will be paralogous within AND among species. Any questions? Please ask in class. No reason to be shy! (It will wake you up. If sleepy BRING COFFEE!)
Reading for Tuesday Sep 30th: Still chapter 4, pages 195-201.
Quiz #4 Q&As
1. Write 4 questions for the next quiz.
Answers will vary
-------------------
Friday, September 26, 2008
Readings for Monday's class
On Monday we will start with chapter 4. Read pages 195-201!
-----------------
-----------------
LAB - GMO gel electrophoresis
In this lab we are simply running the gels to visualize the results of our GMO-testing PCRs. We prepared 2.5% agarose gels as opposed to the most common 1% gels, because the expected fragments are small and very close in size (CMV 35S: ~200 bp; NOS: ~220 bp). In cases like these a denser gel will increase the resolution of the gel by enhancing band separation and making sharper bands.
There were six samples in our gels
Lane 1: Certified non-GMO plant DNA - Plant primers
Lane 2: Certified non-GMO plant DNA - GMO primers
Lane 3: Corn or soy DNA - plant primers
Lane 4: Corn or soy DNA - GMO primers
Lane 5: Positive control DNA - plant primers
Lane 6: Positive control DNA - GMO primers
Lane 7: Ladder
NOTE: Do not worry about questions 2 and 3 on pages 43 and 44 of lab 6 (question 3 is not actually labeled, so skip to question 4).
Here are two of the gels showing the best results. If for some reason your gel didn't come out well, or your PCRs didn't work write that down in your results and use one of these images to compare with your results in the discussion section.
Gel in section 01
Gel in section 02
------------------
Gel in section 01
Gel in section 02------------------
LAB - Genetically Modified Organisms (GMOs)
- Testing for the presence of proteins that the trans-genes produce
- Amplifying a fragment of DNA that contains the trans-gene via PCR
Since we need a lot of fresh tissue and a kit to test for several of the proteins that the most common trans-genes code for, materials that we don't have, we are using the almighty PCR.
Most trans-genes have the 35S promoter from the cauliflower mosaic virus (CMV 35S), and the nopaline synthase (NOS) terminator from Agrobacterium tumefaciens. These sequences come from totally different organisms from that used to isolate the actual trans-gene, but they are recognized by the plant cell machinery (mainly its polymerase), so they can actually produce the product that the gene is intended for. Even if the gene is in the genome, it will NOT work if there are no promoters AND terminators that the plant can recognize. Since there is a number of genes that have been inserted in crops in the U.S., it's easier to go after the promoter and the terminator when using PCR. CMV 35S and NOS have been use in over 85% of the GMOs, so if we have primers to amplify them, we don't have to worry about which gene was inserted (as long as the question doesn't involve knowing the specific gene).
We extracted DNA from corn or soy that we presume have been genetically modified, and from a cerified non-GMO food control provided by Bio Rad, the manufacturers of the kit we use. The master mixes we used include primers for both CMV35S and NOS in the same mix, and in addition to those we are using a separate master mix with primers that will amplify a 455 bp region of the photosystem II (PSII) chloroplast gene, common to most plants. This will be a control to ensure we have successfully extracted DNA, so even non-GM plants should have the band present.
Speaking of PCR, watch the Bio Rad PCR animation NOW...!
Speaking of PCR, watch the Bio Rad PCR animation NOW...!
The gel electrophoresis will be ran the following day.
---------------
Tuesday, September 23, 2008
Genomics and the tree of life
Click on the pic for a full-size imageToday we talked about the diversity of genomes and thier role in recostructing the tree of life. The material is basically covered on pages 15-24 of Alberts' book.
Goals of the topic
- Understand the main reasons for the success of DNA-based life
- Get a basic understanding of how genomes evolve
- Understand how features of genomic evolution shape the tree od life
- Understand the impact of genomics in the field of systematics
We well resume class starting at "New genes are generated from preexisting ones" (p 18).
Quiz #3 - Q&As
1. What is a codon?
In a gene, a series of three nucleotides that encode information that will be ultimately translated into an amino acid in a protein
2. What is translation?
The process in which proteins are synthesized according to RNA sequences carrying over infromation from the DNA
3. What is transcription?
The process in which information found in the DNA is passed onto an RNA molecule
4. What are the three domains in the tree of life?
Archea (Archebacteria), Bacteria (Eubacteria), and Eukaryota
5. What is a gene family?
A group of genes in a genome that descend (via gene duplication) from a single gene
------------------
Friday, September 19, 2008
LABS 3 and 4 - RED and PV92 PCR Gel electrophoreses
Today we finished labs 3 and 4, which we started yesterday.
Lab 3 - Restriction Enzyme Digestions - Gel electrophoresis
We loaded the DNA digestions that we set up yesterday, plus a standard (a.k.a. marker or ladder). Once again, the DNA that was digested (and an un-digested control) came from the lambda bacteriophage, a common phage of E. coli. The enzymes used for the digestion were HindIII, PstI, and EcoRI. The marker was a precut lambda bacteriophage DNA, HindIII.
Methods and results at this point should resemble the ones from lab 2. You need to do a visual estiumation of size of DNA bands, and then use the precut HindIII digestion bands to create a migration distance vs. band size curve in semi-log paper and interpolate (and extrapolate) the information obtained from other samples.
Nice thing about this lab is that STUDENTS actually estimated the band migration distance, as opposed to last week, when the instrctor did it.
Lab 4 - PV92 PCR - Gel electrophoresis
Yesterday after lab, the instructor added the PCR master mix to the human DNA samples donated by the students, and put all the tubes in the thremocycler to perform a PCR to amplify the PV92 locus. Today students ran gel electrphoreses to vizualize the results and determine if they are homozygous (positive or negative) for the Alu sequence insertion in the PV92 DNA segment.
Results will be used to perform a quick and basic bioinformatics exercise using the obtained information in the Allele Server webpage. Access the Dolan DNA Learning Center Gene Almanac and click on 'Resources', then on 'Bioservers' and log on to 'Allele Server'. The username is a-cordoba, and the password is biol217.
Once in there click on 'add data'. On the pull-down menu 'please choose your group' click on 'ONU-biol 217' (which should be the default). The password is biol217 and your number is the one I assigned to you on an e-mail that I sent.
Enter the information (you don't have to enter info about your parents' descent if you don't want to) and click ok. Once all of us have done that we can play with the data and find out a few things of us as a "population".
To VIEW the information, click on 'Manage Groups', and click on 'View' in front of the 'ONU-Biol 217' group.
Click here to find more information about PV92 and the Alu sequence!
----------------
Lab 3 - Restriction Enzyme Digestions - Gel electrophoresis
We loaded the DNA digestions that we set up yesterday, plus a standard (a.k.a. marker or ladder). Once again, the DNA that was digested (and an un-digested control) came from the lambda bacteriophage, a common phage of E. coli. The enzymes used for the digestion were HindIII, PstI, and EcoRI. The marker was a precut lambda bacteriophage DNA, HindIII.
Methods and results at this point should resemble the ones from lab 2. You need to do a visual estiumation of size of DNA bands, and then use the precut HindIII digestion bands to create a migration distance vs. band size curve in semi-log paper and interpolate (and extrapolate) the information obtained from other samples.
Nice thing about this lab is that STUDENTS actually estimated the band migration distance, as opposed to last week, when the instrctor did it.
Lab 4 - PV92 PCR - Gel electrophoresis
Yesterday after lab, the instructor added the PCR master mix to the human DNA samples donated by the students, and put all the tubes in the thremocycler to perform a PCR to amplify the PV92 locus. Today students ran gel electrphoreses to vizualize the results and determine if they are homozygous (positive or negative) for the Alu sequence insertion in the PV92 DNA segment.
Results will be used to perform a quick and basic bioinformatics exercise using the obtained information in the Allele Server webpage. Access the Dolan DNA Learning Center Gene Almanac and click on 'Resources', then on 'Bioservers' and log on to 'Allele Server'. The username is a-cordoba, and the password is biol217.
Once in there click on 'add data'. On the pull-down menu 'please choose your group' click on 'ONU-biol 217' (which should be the default). The password is biol217 and your number is the one I assigned to you on an e-mail that I sent.
Enter the information (you don't have to enter info about your parents' descent if you don't want to) and click ok. Once all of us have done that we can play with the data and find out a few things of us as a "population".
To VIEW the information, click on 'Manage Groups', and click on 'View' in front of the 'ONU-Biol 217' group.
Click here to find more information about PV92 and the Alu sequence!
----------------
Thursday, September 18, 2008
LABS 3 and 4 - RED and PCR
Today we actually started labs 3 and 4, which will be finished tomorrow.
Lab #3
Lab 3 is very similar to Lab 2, except for the fact that this time we ACTUALLY set up the Restriction Enzyme Digestions (REDs), instead of simply running pre-cut DNA in a gel. We added DNA, restriction buffer, and restriction enzymes to a mix, and incubated it for 30 minutes at 37ºC. Why that temperature? The enzymes have been isolated from bacteria that live in mammals and, as we know, tht is our body temperature. That means that the enzymes will be most efficient at that temperature. They will still work at lower temperatures, but they would take a lot longer to do the same job. Since we incubated them at 37º, I stored them in the fridge (4º), where the enzymes won't act almost at all. If we had not incubated the digestions, they should be sitting at room temperature until tomorrow morning.
Tomorrow we will run a gel electrophoresis with such samples and compare them with the ones we ran last week in lab#2. Results should be pretty similar. Yes, you'll have to estimate the size of the DNA contained in the bands, using the visual method and the standard curve using semi-log paper. I'll e-mail you a semi-log paper pdff file in case you want to print some more copies.
Lab 4/5 - PV92 PCR
PV92 is a locus found in chromosome 16 in humans. As you should remember, we all have two copies of each chromosome, so we have two chromosomes 16, and therefore we have two copies of the PV92 locus.
What does PV92 do? Nothing! It does not code for any thing, just as the 95% of the human genome. Only about 5% of our genome actually encodes some sort of product. This locus was named in the old days of karyiotyping, when we didn't really know what different areas stained in a chromosome did. After we sequenced the human genome, we figured out that the names given to many loci didn't actually correspond to a functional gene. PV92 may be a pseudogene (a duplicated gene that lost its function), so for practical purposes we'll consider it a gene..
The nice thing of having a locus that doesn't do any thing at all is that it is not influenced by natural selection. And therefore when we think about mating in primate populations, it is random when it comes down to PV92. So? Well... in population genetics a lot of hypothesis testing is based on the premise of random mating in a given population, so PV92 has been used for a lot of human population genetics studies.
There is another nice thing about PV92. Some people have an Alu sequence inserted in the very middle of the PV92 locus. This Alu insert, an example of an intron (a DNA fragment found in between coding regions of a gene, that doesn't code for any thing and that it's spliced during transcription in functional genes), makes pV92 300 bases longer, which is a difference big enough that it can be visualized in a gel (originally PV92 is about 600 base pairs long).
An Alu sequence is a retrotransposon (a DNA fragment that has been inserted in a location where it doesn't belong via reverse transcription, i.e. mRNA actually writes a sequence into the genome) found throughout primate genomes. There are thousands of copies of Alu sequences found in a single genome.
PCR, the Polymerase Chain Reaction, a method to amplify (make many copies of) specific DNA fragments, is used to generate enough DNA that it can be studied with a variety of thechniques. Using PCR we will amplify our PV92 loci to see if we have the Alu sequence insert in any of our copies of PV92. Each one of us may have 0, 1, or 2 alu inserts in our PV92 copies and we will find out through PCR and gel electrophoresis.
Today we extracted our own DNA. We scraped our cheeks to get some epithelial cells, we neutralized ions (with the InstaGel) that may be used as cofactors by DNases to chop out DNA, and then we bursted our cells open (with the water baths) to extract the DNA from the nuclei.
After lab I added your DNA and mine, plus three positive controls, to a master mix with all the constituents of a PCR: buffer, nucleotides, Taq polymerase, magnesium, and primers (synthesized single stranded oligonucleotide sequences) and put the tubes into the thermocycler. We'll talk about details of how PCR works later.
Tomorrow we'll run a gel electrophoresis to figure out who among us has the Alu insert in his/her PV92 locus, and if it is a single copy or two.
-------------
Lab #3
Lab 3 is very similar to Lab 2, except for the fact that this time we ACTUALLY set up the Restriction Enzyme Digestions (REDs), instead of simply running pre-cut DNA in a gel. We added DNA, restriction buffer, and restriction enzymes to a mix, and incubated it for 30 minutes at 37ºC. Why that temperature? The enzymes have been isolated from bacteria that live in mammals and, as we know, tht is our body temperature. That means that the enzymes will be most efficient at that temperature. They will still work at lower temperatures, but they would take a lot longer to do the same job. Since we incubated them at 37º, I stored them in the fridge (4º), where the enzymes won't act almost at all. If we had not incubated the digestions, they should be sitting at room temperature until tomorrow morning.
Tomorrow we will run a gel electrophoresis with such samples and compare them with the ones we ran last week in lab#2. Results should be pretty similar. Yes, you'll have to estimate the size of the DNA contained in the bands, using the visual method and the standard curve using semi-log paper. I'll e-mail you a semi-log paper pdff file in case you want to print some more copies.
Lab 4/5 - PV92 PCR
PV92 is a locus found in chromosome 16 in humans. As you should remember, we all have two copies of each chromosome, so we have two chromosomes 16, and therefore we have two copies of the PV92 locus.
What does PV92 do? Nothing! It does not code for any thing, just as the 95% of the human genome. Only about 5% of our genome actually encodes some sort of product. This locus was named in the old days of karyiotyping, when we didn't really know what different areas stained in a chromosome did. After we sequenced the human genome, we figured out that the names given to many loci didn't actually correspond to a functional gene. PV92 may be a pseudogene (a duplicated gene that lost its function), so for practical purposes we'll consider it a gene..
The nice thing of having a locus that doesn't do any thing at all is that it is not influenced by natural selection. And therefore when we think about mating in primate populations, it is random when it comes down to PV92. So? Well... in population genetics a lot of hypothesis testing is based on the premise of random mating in a given population, so PV92 has been used for a lot of human population genetics studies.
There is another nice thing about PV92. Some people have an Alu sequence inserted in the very middle of the PV92 locus. This Alu insert, an example of an intron (a DNA fragment found in between coding regions of a gene, that doesn't code for any thing and that it's spliced during transcription in functional genes), makes pV92 300 bases longer, which is a difference big enough that it can be visualized in a gel (originally PV92 is about 600 base pairs long).
An Alu sequence is a retrotransposon (a DNA fragment that has been inserted in a location where it doesn't belong via reverse transcription, i.e. mRNA actually writes a sequence into the genome) found throughout primate genomes. There are thousands of copies of Alu sequences found in a single genome.
PCR, the Polymerase Chain Reaction, a method to amplify (make many copies of) specific DNA fragments, is used to generate enough DNA that it can be studied with a variety of thechniques. Using PCR we will amplify our PV92 loci to see if we have the Alu sequence insert in any of our copies of PV92. Each one of us may have 0, 1, or 2 alu inserts in our PV92 copies and we will find out through PCR and gel electrophoresis.
Today we extracted our own DNA. We scraped our cheeks to get some epithelial cells, we neutralized ions (with the InstaGel) that may be used as cofactors by DNases to chop out DNA, and then we bursted our cells open (with the water baths) to extract the DNA from the nuclei.
After lab I added your DNA and mine, plus three positive controls, to a master mix with all the constituents of a PCR: buffer, nucleotides, Taq polymerase, magnesium, and primers (synthesized single stranded oligonucleotide sequences) and put the tubes into the thermocycler. We'll talk about details of how PCR works later.
Tomorrow we'll run a gel electrophoresis to figure out who among us has the Alu insert in his/her PV92 locus, and if it is a single copy or two.
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Tuesday, September 16, 2008
A first glance at molecules within the cell
Today we covered mainly material between pages 1 and 8 in the textbook. We talked about the universality of the hereditary information code (mostly DNA), what are its building blocks, how it replicates within the cell, it is transcribed into RNA, and how it is translated into proteins. In a few words, we went over how a gene is expressed.
We talked at a very basic level, and some of these topics will be revisited in the future and explored further.
On Monday (Sep 22): We'll talk about the secondary structure of RNA, some details of how it is translated into proteins, and what are some of the most common functions of such molecules.
We'll also talk about the implication of studying DNA at the genomic level (again, a very basic, introductory talk).
Reading material: Pages 15-24 of the textbook.
Today's quiz Q&As
1. What is templated polymerization?
The specific mechanism through which DNA replicates, using a strand of DNA as a template to the other
2. What are the two main processes that result in the synthesis of a protein that is encoded in the DNA?
Transcription and translation
3. What is a polypeptide?
A chain of amino acids (All proteins are polypeptides, not all polypeptides are proteins. Not to be confused with polynucleotide, a chain of nucleotides)
4. What is a codon?
A triplet of nucleotides that act as a coding unit for a specific amino acid, or as signals to start and stop the transcription of a gene
5. What is the main function of proteins in a cell?
They are mainly catalysts for a huge veriety of reactions.
(Yes, they are also the building blocks, and agents of cellular organelle repair, but most of them are catalysts of some sort)
3. What is a polypeptide?
A chain of amino acids (All proteins are polypeptides, not all polypeptides are proteins. Not to be confused with polynucleotide, a chain of nucleotides)
4. What is a codon?
A triplet of nucleotides that act as a coding unit for a specific amino acid, or as signals to start and stop the transcription of a gene
5. What is the main function of proteins in a cell?
They are mainly catalysts for a huge veriety of reactions.
(Yes, they are also the building blocks, and agents of cellular organelle repair, but most of them are catalysts of some sort)
Monday, September 15, 2008
The importance of Molecular Biology - topics for review papers
Today we had our first quiz (using the bluebooks), talked a little about the importance of molecular biology, and browsed over a brochure called "Genomics and its impact on science and society", put together by the U.S. Department of Energy Office of Science (see me if you didn't get one). The brochure has an interesting bulleted list on page 7 about applications of molecular biology in different fields.
Also, students teamed up in pairs to propose potential topics for their lieterature review paper in some aspect of molecular biology. We went over some of the poropsoed topics and next week I'll meet with each team in my office (preferably during office hours. Make an appointment so you don't have to wait if I am meeting with another team) to go over your potential topics, your motivation for choosing them, your justification, and to select one of them for your paper.
Q&A's for today's quiz:
1. What are the building blocks of a nucleotide?
Phosphate, sugar, and base
2. What are the processes that result in the synthesis of a protein encoded in the DNA?
Transcription and translation
3. How are new genes generated? (a brief generic explanation. No need to list the specific processes)
My modification of a pre-existing gene (e.g. mutation, duplication, segment shuffling, or horizontal transfer)
4. Mention one way in which the function of a gene can be revealed
By studying its sequence and comparing it to previsouly known ones, by studying the effect of mutations, or by knocking-out genes
5. What is a model organism?
An easy to breed, intensively studied genetically, and therefore well known organism. Conclusions from its study can be extrapolated to other organisms (e.g., the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, the mouse Mus musculus, or the thale cress Arabidopsis thaliana)
Also, students teamed up in pairs to propose potential topics for their lieterature review paper in some aspect of molecular biology. We went over some of the poropsoed topics and next week I'll meet with each team in my office (preferably during office hours. Make an appointment so you don't have to wait if I am meeting with another team) to go over your potential topics, your motivation for choosing them, your justification, and to select one of them for your paper.
Q&A's for today's quiz:
1. What are the building blocks of a nucleotide?
Phosphate, sugar, and base
2. What are the processes that result in the synthesis of a protein encoded in the DNA?
Transcription and translation
3. How are new genes generated? (a brief generic explanation. No need to list the specific processes)
My modification of a pre-existing gene (e.g. mutation, duplication, segment shuffling, or horizontal transfer)
4. Mention one way in which the function of a gene can be revealed
By studying its sequence and comparing it to previsouly known ones, by studying the effect of mutations, or by knocking-out genes
5. What is a model organism?
An easy to breed, intensively studied genetically, and therefore well known organism. Conclusions from its study can be extrapolated to other organisms (e.g., the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, the mouse Mus musculus, or the thale cress Arabidopsis thaliana)
Friday, September 12, 2008
LABS - Micropipettes, RED anlyses and gel electrophoreses...
THURSDAY, SEPT 11th
We had an introduction to the lab and I handed out lab notebooks, lab manuals, and 'BlueBooks' (examination notebooks for lecture quizzes).
Working in the lab
You will work in pairs, but lab reports are to be handed in individually. Results for both team members should be the same, but the discussion (see below) should be different. You are allowed to discuss results with any one in the lab, including the instructor, but write your lab discussion on your own.
Lab reports are to be handed in at the end of the lab or the following week, depending on the exercise. You must turn in the copy page of the lab notebook, and keep the original. Remember to put the 'flap' of the back cover in between pages, so you don't end up writing on the next page duplicate. Just as in a checkbook.
Here's what I expect in terms of lab reports:
1. Title and date
2. Introduction
Make it short (no more than half a page) and in your own words. No need to copy straight from the book.
3. Materials
4. Methods
Again, in your own words. I just want to know that you understand what you'll do in the lab that day.
5. Results
Straightforward. Just what you observe, measure, etc. No un-specific comments. Save those for the discussion.
6. Discussion
Although you are allowed, and even encouraged, to informally discuss your results with any one, write this section on your own. If you are not use to write scientifically on your own, it's time to begin doing so.
7. Questions
Most labs have specific questions that you must answer. They will be related to the lab topic, although not necessarily directly related to what you are doing in the exercise.
No need to re-write the question in the notebook. Just mention the page number and if the questions in that page are not numbered the first one is assumed to be number one and so on. The first question in a new page will be #1 in that other page.
Lab exercise #1
Using micropipettes:
A lab designed for those of you without previous experience in the use of a micropipette, one of the most common instruments in molecular biology. They are used especially when you have to measure accurately volumes under 1mL, although some micropipettes can measure up to 10mL. The unit of measure in most micropipettes (including all of those we used in the lab) is a 1000th of a mL, or a μL (microliter). A micropipette is nothing but a sophisticated pipette. It doesn't any thing that a glass pipette or a graduated cylinder doesn't do, except for dealing with REALLY small volumes (smaller than a rain drop) accurately.
Micropipettes have different ranges of measurement and use different plastic tips, which in general are color coded. Informally the micropipettes are named according to the range of volumes they measure:
0.5-10 μL: "P-10", clear or "white" tips
20-200 μL: "P-200", yellow tips
100-1000 μL: "P-1000", blue tips
The exercise consisted in measuring volumes of colored liquids (we used food coloring) so you could get used to the functioning of the micropipettes.
FRIDAY, SEPT 12th
Lab exercise #2
An introduction to Restriction Analysis
Restriction enzymes (or restriction endonucleases) cleave (cut) DNA in a very specific fashion. They have a unique nucleotide sequence at which they cut a DNA molecule. A particular restriction enzyme will cleave DNA at that recognition sequence and nowhere else. The recognition sequence is often a six base pair palindromic sequence but others recognize four or even eight base pair sequences. The act of exposing DNA to a restiction enzyme is known as a 'digestion', and hence the acronym RED: Restriction Enzyme Digestion.
Restriction enzymes can also differ in the way they cut the DNA molecule. Some enzymes cut in the middle of the recognition sequence, resulting in a blunt end. Other enzymes cleave in a staggered fashion, resulting in DNA products that have short single-stranded overhangs (usually two or four nucleotides) at each end. These are often called cohesive ends (also known as "sticky" ends), as these single-stranded overhangs could potentially come together again through complementary base-pairing.
A common use for restriction enzymes is to generate a "fingerprint" of a particular DNA molecule. Because of the sequence specificity of restriction enzymes, these enzymes can cut DNA into discrete fragments which can be resolved by gel electrophoresis. This pattern of DNA fragments generates a "DNA fingerprint," and each DNA molecule has its own fingerprint. Other restriction enzymes can be used to further characterize a particular DNA molecule. The location of these restriction enzyme cleavage sites on the DNA molecule can be compiled to create a restriction enzyme map. These maps are very useful for identifying and characterizing a particular DNA plasmid or region.
(The restiction enzyme information above has been modified from the Restriction Enzyme Analysis of DNA web page, from the University of Arizona Biotech project.)
We didn't actually perform a RED, but we did use Bacteriophage Lambda (a virus that 'feeds' [kills to hijack its cell machinery] on bacteria) DNA that was alredy digested with three different restriction enzymes: EcoRI, HindIII, and PstI. We also had an un-digested sample as a control. By having these DNA samples were able to run a gel electrophoresis to observe the band pattern resulting from each RED and estimate the size of the DNA strands in each band in the gel. In this lab we are actually doing a crude restriction enzyme analysis.
Most of the gels we ran in the lab didn't show any results. Students didn't do any thing wrong, and the lack of results was explained because the gels were either not stained, or were too old and the gel stain degraded. Below I am posting a gel picture from each lab section (one gel per section worked really well). You can download the picture to estimate the band sizes as explained in the lab manual. To generate the standard curve see the data that I took from each one of the gels. You should use the gel that worked in YOUR lab section.
In both gels the lanes are:
1. Un-cut bacteriophage lambda DNA
2. PstI RED
3. EcoRI RED
4. HindIII RED
Distances (in mm) bands have migrated from the origin:
Number of bands may be different on each table and you may not see as many bands in the picture. These are caveats of molecular work: Not all imaging systems are sensitive enough and you may see more bands if you examine the gel with the naked eye. The staining in the gel may vary as well, plus samples that have been diluted with too much loading dye or have not been properly loaded into the gel may appear weaker so the observer may miss the fainter bands.
All these are things you may consider in your discussion. I am sure we'll run into more similar problems (or new ones) in the next few weeks.
------------
Micropipettes have different ranges of measurement and use different plastic tips, which in general are color coded. Informally the micropipettes are named according to the range of volumes they measure:
0.5-10 μL: "P-10", clear or "white" tips
20-200 μL: "P-200", yellow tips
100-1000 μL: "P-1000", blue tips
The exercise consisted in measuring volumes of colored liquids (we used food coloring) so you could get used to the functioning of the micropipettes.
FRIDAY, SEPT 12th
Lab exercise #2
An introduction to Restriction Analysis
Restriction enzymes (or restriction endonucleases) cleave (cut) DNA in a very specific fashion. They have a unique nucleotide sequence at which they cut a DNA molecule. A particular restriction enzyme will cleave DNA at that recognition sequence and nowhere else. The recognition sequence is often a six base pair palindromic sequence but others recognize four or even eight base pair sequences. The act of exposing DNA to a restiction enzyme is known as a 'digestion', and hence the acronym RED: Restriction Enzyme Digestion.
Restriction enzymes can also differ in the way they cut the DNA molecule. Some enzymes cut in the middle of the recognition sequence, resulting in a blunt end. Other enzymes cleave in a staggered fashion, resulting in DNA products that have short single-stranded overhangs (usually two or four nucleotides) at each end. These are often called cohesive ends (also known as "sticky" ends), as these single-stranded overhangs could potentially come together again through complementary base-pairing.
A common use for restriction enzymes is to generate a "fingerprint" of a particular DNA molecule. Because of the sequence specificity of restriction enzymes, these enzymes can cut DNA into discrete fragments which can be resolved by gel electrophoresis. This pattern of DNA fragments generates a "DNA fingerprint," and each DNA molecule has its own fingerprint. Other restriction enzymes can be used to further characterize a particular DNA molecule. The location of these restriction enzyme cleavage sites on the DNA molecule can be compiled to create a restriction enzyme map. These maps are very useful for identifying and characterizing a particular DNA plasmid or region.
(The restiction enzyme information above has been modified from the Restriction Enzyme Analysis of DNA web page, from the University of Arizona Biotech project.)
We didn't actually perform a RED, but we did use Bacteriophage Lambda (a virus that 'feeds' [kills to hijack its cell machinery] on bacteria) DNA that was alredy digested with three different restriction enzymes: EcoRI, HindIII, and PstI. We also had an un-digested sample as a control. By having these DNA samples were able to run a gel electrophoresis to observe the band pattern resulting from each RED and estimate the size of the DNA strands in each band in the gel. In this lab we are actually doing a crude restriction enzyme analysis.
Most of the gels we ran in the lab didn't show any results. Students didn't do any thing wrong, and the lack of results was explained because the gels were either not stained, or were too old and the gel stain degraded. Below I am posting a gel picture from each lab section (one gel per section worked really well). You can download the picture to estimate the band sizes as explained in the lab manual. To generate the standard curve see the data that I took from each one of the gels. You should use the gel that worked in YOUR lab section.
In both gels the lanes are:
1. Un-cut bacteriophage lambda DNA
2. PstI RED
3. EcoRI RED
4. HindIII RED
Gel for lab section 01
(click on pic for full-size image)
(click on pic for full-size image)
Gel for lab section 02
(click on pic for full-size image)
(click on pic for full-size image)
Distances (in mm) bands have migrated from the origin:
Gel section 01
Gel section 2
Number of bands may be different on each table and you may not see as many bands in the picture. These are caveats of molecular work: Not all imaging systems are sensitive enough and you may see more bands if you examine the gel with the naked eye. The staining in the gel may vary as well, plus samples that have been diluted with too much loading dye or have not been properly loaded into the gel may appear weaker so the observer may miss the fainter bands.
All these are things you may consider in your discussion. I am sure we'll run into more similar problems (or new ones) in the next few weeks.
------------
Introducing the BIOL 217 blog! (Intro to Molecular Biology)
Welcome to the Introduction to Molecular Biology (BIOL 217) blog!
This blog is meant to be a reference for all students in the class, to keep track of what we do in lecture and in the lab, and when we do it. My goal is to have an entry per class and lab, which may vary from a few lines to a few pages of text and/or images.
Comments are allowed, so feel free to write something you may consider ncessary to share. I am also open to comments and criticisms (constructive, please!) that may help me run a better blog. This tool will allow me to do several things that I would use a WebCT site for, but in an easier way (and more fun for me). What we cannot do, that WebCT allows us to do, is keep track of grades, and upload/download files (since I don't have access o the actual server). If some one knows a way around this issue, I'll be happy to learn!
If there is popular demand for a WebCT site, I'll set it up. In the mean time, any necessary materials can be copied from the p-drive.
Since I have not had a blog for a class with a lab component before, in a way this one is an experiment. Hopefully it will work, but that's why I need feed back. Please let me know what you would like to see posted here, and in what way. Are there any elements should I add or delete?
Just write a comment or send me an e-mail: a-cordoba@onu.edu.
Cheers to every one...
Alonso
--------------
This blog is meant to be a reference for all students in the class, to keep track of what we do in lecture and in the lab, and when we do it. My goal is to have an entry per class and lab, which may vary from a few lines to a few pages of text and/or images.
Comments are allowed, so feel free to write something you may consider ncessary to share. I am also open to comments and criticisms (constructive, please!) that may help me run a better blog. This tool will allow me to do several things that I would use a WebCT site for, but in an easier way (and more fun for me). What we cannot do, that WebCT allows us to do, is keep track of grades, and upload/download files (since I don't have access o the actual server). If some one knows a way around this issue, I'll be happy to learn!
If there is popular demand for a WebCT site, I'll set it up. In the mean time, any necessary materials can be copied from the p-drive.
Since I have not had a blog for a class with a lab component before, in a way this one is an experiment. Hopefully it will work, but that's why I need feed back. Please let me know what you would like to see posted here, and in what way. Are there any elements should I add or delete?
Just write a comment or send me an e-mail: a-cordoba@onu.edu.
Cheers to every one...
Alonso
--------------
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