| Life Sciences 3 Introduction to Molecular Biology | ||||||||
| Lab Modules - Concepts and Skills | ||||||||
| LAB 1 - Pipetter Exercise & Analysis of Protein Size Using SDS-PAGE | ||||||||
| In this lab students are going to get to know the most commonly used instrument in molecular biology – the micropipetter. Students will learn the proper procedure for operating pipetters during this exercise. Then they use the pipetters to conduct an SDS-PAGE experiment. One of the basic ways to understand a protein is to know its mass. There are a few methods to learn the mass of a protein. In this exercise students will use gel electrophoresis to estimate the mass of the subunits of an unknown protein. In conjunction with information gathered from other methods, the number of subunits of the unknown protein also will be determined. | ||||||||
| Concepts – | ||||||||
| Understanding protein structure | ||||||||
| Understanding protein size estimation methods | ||||||||
| Skills – | ||||||||
| Proper use of micropipetters | ||||||||
| Loading an SDS-PAGE gel | ||||||||
| Creating a standard curve in Excel | ||||||||
| Determining the size of an unknown polypeptide using a standard curve | ||||||||
| LAB 2 - Biochemical Assay of β-Galactosidase Activity | ||||||||
| Beta-galactosidase is an enzyme that breaks down lactose into two monosaccharides. Students will learn to measure the activity of the enzyme by measuring the rate at which products appear and the time required for this enzyme to be synthesized. | ||||||||
| Concepts – | ||||||||
| Understanding the lac operon and its function | ||||||||
| Using an assay to indirectly calculate enzymatic activity | ||||||||
| Skills – | ||||||||
| Proper use of micropipetters | ||||||||
| Correct handling and disposal of bacterial cells | ||||||||
| Use of the spectrophotometer, including using the correct blank | ||||||||
| LAB 3 - DNA Isolation & Amplification | ||||||||
| Polymerase chain Reaction (PCR) is a technique that allows scientists to make many copies of DNA from a small sample. In this lab, students will isolate DNA from cheek cells and prepare the sample for PCR. The samples will be amplified and sent out for sequencing for use in lab 5. Good primer design is a vital part of developing a PCR protocol, so students will be asked to practice creating a primer pair in the primer design exercise. | ||||||||
| Concepts – | ||||||||
| Primer design (by hand and using Primer-3) | ||||||||
| Mitochondria and maternal lineage | ||||||||
| PCR for DNA amplification | ||||||||
| How primers work | ||||||||
| 5' to 3' | ||||||||
| Skills – | ||||||||
| Proper use of micro-pipetters | ||||||||
| Chelex extraction of DNA from cheek cells | ||||||||
| Use BLAST to check primer uniqueness | ||||||||
| LAB 4 - Agarose Gel Electrophoresis & Molecular Clocks | ||||||||
| In this lab students will receive a portion of your amplified DNA sample from lab 3. They will check for PCR product in two ways: by visualizing the product on an agarose gel and by checking the concentration using the spectrophotometer. Students also will learn how to use BLAST, ClustalX and Mega4, programs to be used in the analysis of individual sequences in lab 5. | ||||||||
| Concepts – | ||||||||
| Understand qualitative and quantitative measures of PCR success | ||||||||
| Understand MCRA and how to read phylogenetic trees | ||||||||
| Understand the concept of the molecular clock | ||||||||
| Skills – | ||||||||
| Proper use of micropipetters | ||||||||
| Load and run an agarose gel | ||||||||
| Use the spectrophometer to determine DNA concentration | ||||||||
| Use BLAST to align two DNA sequences | ||||||||
| Estimate a divergence rate for a set of sequences | ||||||||
| Align multiple DNA sequences in ClustalX | ||||||||
| Draw a phylogenetic tree in Mega4 | ||||||||
| LAB 5 - Sequence Analysis and Maternal Lineages | ||||||||
| In this lab students will receive their mitochondrial DNA sequence, which was sequenced off site after the PCR amplification. Students will compare it to many other sequences, an analysis that will allow them to determine their maternal lineage based on human migration patterns. Students will annotate their sequence and re-visit the programs used in lab 4 to create phylogenetic trees with their own sequence. | ||||||||
| Concepts – | ||||||||
| Understand dideoxy sequencing method | ||||||||
| Understand human migration patterns and haplogroups | ||||||||
| Interpret data obtained from BLAST | ||||||||
| Interpret a phylogenetic tree | ||||||||
| Skills – | ||||||||
| Use Chromas to clean up a DNA sequence | ||||||||
| Use BLAST to search for a haplogroup | ||||||||
| Annotate a sequence | ||||||||
| Align sequences in ClustalX | ||||||||
| Draw a phylogenetic tree using Mega4 | ||||||||
| LS Laboratory Website | ||||||||
| Information on class, section and room schedule can be found here: | ||||||||
| http://lslab.lscore.ucla.edu | ||||||||