Laboratory DNA Science: An Introduction to Recombinant DNA Techniques and Methods of Analysis (1996) Bloom, Freyer, & Micklos (eds.), The Benjamin/Cummings Publishing Company, Inc., Menlo Park, CA
You will need to supply your own laboratory textbook, a laboratory notebook (a standard lab notebook is recommended, but any form that you can keep organized and turn-in to the TA will be acceptable), 2-3 Sharpie(TM)-style permanent, water-resistant, extra fine point lab markers, laboratory goggles, and at least 19-pairs of powder-free gloves (one pair for each lab class).
There will be no quizzes. Your grade is based upon class participation and your laboratory write-ups. However, your participation grade is based upon, in part, your being prepared when you come to class. You are expected to have read the exercises prior to the laboratory meeting. If at any point it appears that the students in the class are not preparing beforehand, the TA may present an unannounced quiz.
There will be no final exam in this class.
Ten points are available per lab, but points may be deducted for less than full participation, such as arriving late, not applying yourself to the task during the lab, or leaving early.
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Chapter |
Description |
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1
2
04/01/02 |
Measurements, micropipetting, and sterile techniques. Introduction to micropipetting and sterile pipetting techniques used throughout the course with special attention to manipulations of very small liquid volumes Bacterial culture techniques. Focus on the culture techniques that will be used throughout the course |
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3
04/03/02 |
DNA restriction and manipulation. Introduction to genotypic analysis of DNA using restriction endonucleases and gel electrophoresis. |
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5
04/08/02 |
Rapid colony transformation of E. coli with plasmid DNA. Transformation of competent E. coli cells with a foreign gene (ampicillin resistance) |
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6
04/10/02 |
Purification and identification of plasmid DNA. Small-scale protocol to purify plasmid DNA from transformed E. coli and confirmation of pAMP transformation into E. coli from exercise 5 |
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7
04/15/02 |
Recombination of antibiotic-resistance genes. Construction of a recombinant plasmid that contains both ampicillin- and kanamycin-resistance genes |
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8
04/17/02 |
Transformation of E. coli with recombinant DNA. Classic procedure for the preparation of competent cells and transformation with pAMP/pKAN plasmids |
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9
04/22/02 |
Replica plating to identify mixed E. coli populations. Use of replica plating to distinguish between singly- and doubly-resistant colonies growing on "Lig" LB/amp and "Lig" LB/kan plates |
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10
04/24/02 |
Purification and identification of recombinant plasmid DNA. Plasmid mini-preparation of pAMP/pKAN recombinants followed by restriction endonuclease analysis |
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10
04/29/02 |
Purification and identification of recombinant plasmid DNA, cont. Complete analysis of recombinants
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13
05/01/02 |
Southern hybridization of l DNA. Restriction endonuclease digestion and electrophoresis of l DNA |
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13
05/06/02 |
Southern hybridization of l DNA, cont. Southern transfer of cleaved l DNA and probe preparation |
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13 18
05/08/02 |
Southern hybridization of l DNA, cont. Non-radioactive probe detection Amplification and purification of a l DNA fragment. Amplification of a l DNA fragment from a genomic library |
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18
05/13/02 |
Amplification and purification of a l DNA fragment, cont. Electrophoresis of a l PCR product and comparison to a BamHI/HindIII digest of l DNA |
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20
05/15/02 |
Transformation of E. coli with PCR product. Ligation of PCR product into pUC19 followed by transformation with blue/white screen |
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20 20+
05/20/02 |
Transformation of E. coli with PCR product, cont. Recovery and analysis of transformants Transformation of S. cerevisiae with PCR product. Transformation of ligated PCR product into S. cerevisiae |
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20+ 22
05/22/02 |
Transformation of S. cerevisiae with PCR product, cont. Recovery of yeast transformants Detection of a specific gene sequence in the human genome. PCR amplification is used to detect the presence of a specific gene sequence in the human genome. |
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--- 05/27/02 |
Memorial Day. University closed. |
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20+ 22
05/29/02 |
Transformation of S. cerevisiae with PCR product, cont. Replica-plating of yeast transformants Detection of a specific gene sequence in the human genome. Electrophoresis and analysis of gene amplification |
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20+
06/03/02 |
Transformation of S. cerevisiae with PCR product, cont. Functional analysis of yeast transformants |
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06/05/02 |
Conclusions, wrap-up, and laboratory clean-up. This is an opportunity for students to finish loose ends of their final experiments, turn in their notebooks, and to clean-up their work areas. Students should properly dispose of all plates, reagents, etc. unless otherwise specified. |
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06/10/02 thru 06/14/02 |
Final Exams |