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.
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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.
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