Cell and Molecular Biology Laboratory
Dr. Brad Stith, Biology Dept. Spring 2006 office hours 2:30 to 3:30 pm M and W although feel free to stop by anytime (calling ahead would be best). Office: 3402 NC, tele: 303 556 3371.
This new lab course will cover selected topics from General Cell Biology 3611- only in more depth through lecture and hands-on lab experience. We will use the same textbook (the newest edition of World of the Cell by Becker et al.).
For
the Syllabus, click here.
Key for cell lab exam 1 (average of 92 out of 119 pts or 77%): click here
Tentative Topics for new Cell and Molecular Biology laboratory:
1. Basic lab skills. Lab safety, glassware, use of pipettors, scales, making of buffers, use of PubMed, basic Student’s t Test statistics, Bradford protein assay, etc. Students will write own lab notebook; use 3 ring binder; add topics to it as you go. Graded at the middle and end of the class. Use a table of contents in the front. List of steps in various techniques/methods (e.g., protein assay).
2. Protein Folding lab– use science computer lab. Use of Mage program to view components of protein structure.
3. Microscopy; immunohistology and electron microscopy.
4. Cell membrane lab; Use of Neuro computer program in science computer lab.
5. Membrane potential lab; using iWorx probe and
amplifiers connected to computers (recording and analysis software comes with
amplifier). Record potential and alter it through use of chemicals. The use of
Xenopus as a research animal is discussed.
6. Cell culture lab; where students make buffers and culture the Xenopus oocyte.
7. Cell signaling lab. After culturing the oocyte, add hormones to induce meiotic cell division. Insulin vs. progesterone action, ERK2/map kinase (using an ELISA), and the tyrosine kinase receptor pathway leads to the activation of cdc2 kinase.
8. Fertilization; record Em, use ELISA to study process. Video and still digital photography to record events. Continuation of Cell Division following mitosis in early embryo.
9. Cell organelles; mitochondria. Isolate Xenopus liver mitochondria with Centrifugation and add substrates and use spectrophotometer to follow reactions. Effect of metformin on Xenopus mitochondria.
10. Computer Lab on molecular biology; exercises on BLAST, Clustal analysis (held in science computer lab)
11. RT- PCR; isolation of mRNA, cDNA preparation and PCR.
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Intro Lecture for Cell Mol Bio Lab
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SigmaPlot Exercise (BRING YOUR CALCULATOR)
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MAKING SOLUTIONS, BASIC CELL CULTURE
Units of measurement in science; click here to visit this web site\
A web site with buffer calculators, molecular weight calculators, etc. CLICK HERE.
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Checking the concentration of your insulin solution: USE OF THE SPECTROPHOTOMETER
for more detail on a Spectrophotomter, visit this web site
Adding Insulin to your cells:
how to
use of micropipettors-read, print and understand the info here
Questions for Making Solutions
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MAP KINASE LAB (click here for PowerPoint Lecture)
Dr. Donald Slish (SUNY Plattsburgh) has animated the receptor tyrosine kinase-map kinase path;
click here to go to the web site.
A recent story on breast cancer and map kinase;
click here to view it.
Here is another web site that has an animation of the receptor tyrosine kinase/map kinase path. This site also has the NOTCH pathway (which I discuss in Devel Biol or Cell Signaling but typically not in Cell Biology 3611). Click here to go the web site of "Biocreations" --a company that makes science animations.
There is a site on receptor tyrosine kinases that is sponsored by a drug company. This drug company site centers in on the epidermal growth factor (EGF) receptor and they note that drugs that inhibit this receptor kinase will stop cancer. For example, AstraZeneca has a drug called Iressa, Novartis has Gleevec and Genentech has Herceptin. Click here.
There is an interesting clinical discussion of fighting cancer by turning off the tyrosine kinase path. Among other drugs, the article noted below discusses the new drug Erbitux (by ImClone and Bristol-Myers Squibb Co)--it is an antibody to epidermal growth factor receptor (the receptor is a tyrosine kinase). This is the drug that was associated with the the stock scandal that caused Martha Stewart trouble! At first, they thought it did not work and so the stock went downhill; now they think that it does work. For the article, see: http://www.411cancer.com/syndication/veContent.jsp?ArticleID=egfr_02&ArticleTypeID=CTON
Mutations in Fibroblast Growth Factor (FGF) receptor tyrosine kinases and the map kinase path have been associated with dwarfism,
Pfeiffer syndrome (a malformation syndrome characterized by limb defects and by
the premature fusion of the cranial sutures -craniosynostosis- that results in
abnormal skull and facial shape), Crouzon Syndrome (have craniosynostosis, but
have normal limbs), Jackson-Weiss syndrome (a syndrome of craniofacial
malformation and foot abnormalities: enlarged great toes and the coalescence of
the tarsals and metatarsals), and Apert Syndrome (a set of malformations
involving craniosynostosis and severe syndactyly or fusion of digits)(Click here to view
the site).
Map kinase (ERK) ELISA procedure We will add hormone (progesterone or insulin) to Xenopus oocytes, and look for any activation of map kinase (ERK). We record the activation of this kinase by recording the amount of ERK with phosphate on it (using an ELISA: a specific antibody binds only to the activated and phosphorylated ERK; the antibody is attached to an enzyme that makes a blue color; more blue color, more active ERK).
Go to this web site for an animation of the ELISA assay; click here
Look at this web site for a description of ELISA assays: click here
view this other animation of the ELISA assay: click here
French paper discussing an early activation of ERK by progesterone; click here
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MEMBRANE POTENTIAL AND NERNST equation PowerPoint lecture- click here
Neurons in Action: resting membrane potential and the Nernst Equation; click here for Neuro Lab
Membrane potential recording lab; our lab protocol
Detail on making and setting up the microelectrode; click here
Original paper reporting Acetylcholine depolarizes Xenopus oocytes; click here
Here is a web site on acetylcholine. When you read it, remember that Xenopus oocytes have the muscarinic M3 subtype of acetylcholine receptor: http://www.chemistry.emory.edu/justice/chem190j/feb15.htmr
A web site on Xenopus electrophysiology; click here
Another lab manual for Xenopus electrophysiology: click here
A paper on Xenopus electrophysiology (comparing it to another frog); click here
For those mathematically inclined, look at this paper on the Nernst equation; click here
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MITOCHONDRIA lab PowerPoint Presentation; click here
Mitochondrial lab Protocol
Protease inhibitor cocktail (does not contain EDTA which harms mitocondria); click here
Discussion of Cell homogenization and Bradford Protein assay; click here
Mitochondrial function:Click here to view the Quicktime animation on chemiosmosis (VERY large file; 14 MB).
Why learn about mitochondria? Bad mitochondrial function may lead to diabetes; click here for the link to the article
Due to the 1974 Nobel Prize on cell organelles, the Nobel Prize web site has a game to play on cell organelles, the ultracentrifuge (used to separate organelles for study), and the TEM (used to observe cell parts); click here to play the game.
This new web site (Virtual Cell Animation Collection) has one of the best animations of electron transport and another on ATP synthase (the flow of electrons turns the synthase like a turbine to make ATP-- cool!) -- CLICK HERE
Here is a web site with a great animation of the electron transport chain and
chemiosmosis.
Click here.
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RT-PCR PowerPoint Lecture
RT-PCR protocol
Second Lab period: agarose electrophoresis, protocol
Read only the first pages and pertinent sections; here is the 30 page manual for the RNA prep kit; click here
I suggest that you read the 4 page manual for the One-Step SuperScript RT-PCR system; click here
Reading the original literature; paper on RT PCR of phospholipase C gamma from mouse; click here
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FERTILIZATION LAB; click here for PowerPoint presentation
See Dr. Stith's home page (www.cudenver.edu/~bstith) to view videos on fertilization
Fertilization Protocol, click here
Procedure for Still Camera: click here
video from Apr 19 2006 fertilization showing cleavage; click here
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BIOINFORMATICS; click here for lecture
Bioinformatics Protocol; click here
Second bioinformatics Protocol; click here
Only after second bioinform protocol finished: Lecture on interpretation of Ras information and structure; click here
Right click on the following link and save this file to your computer desktop where you can find it:: c14Recp.kin
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