Davidson College Molecular Biology Lab Schedule 2002

This work is sponsored in part through an NSF-ILI grant.

Laboratory Schedule: Molecular Biology

This page gives you a weekle outline of what we will do in lab each week as we use PCR to clone five yeast IDH genes, express them in E. coli, determine exhamine hybridization strengencies at the DNA level, affinity purify the proteins, and perform IDH enzyme assays with your recombinant proteins. You MUST keep a lab notebook. To learn what I expect in your notebook, click here.

If the method is followed by a company's product, then we will use their reagents and protocols which will be duplicated for you. Otherwise, follow the links to see detailed protocols.

Week Begining - Procedures

Jan. 15/17: Start at the beginning (practice calculations #1 and #2)

1) We will watch a safety video and make stock solutions that will be used throughout the semester. In addition, we will practice pipetting because in molecular biology, you MUST be good at pipetting.

Jan. 22/24: Purify genomic DNA and design PCR primers

1) We will begin by purifying yeast genomic DNA as our template for PCR.
Qiagen DNeasy Kit #69104

2) Determine the concentration of your gDNA.

3) Find all 5 genes, restriction sites for each gene, expression plasmid sequence, design PCR primers to produce in-frame fusion protein with His₆ epitope and affnity purification tag.
Use yeast genomic web pages and Qiagen QIAexpress UA cloning vector map.

Jan. 29/31: Continue to Design PCR Primers, Make Web Pages

1) You will need to finalize the design of your PCR primers and show them to Dr. C.

2) Finish collecting any information about these 5 genes and make your web sites.
Netscape Composer and Macromedia Dreamweaver. Also use Snapz Pro² for screen shots.

3) Design PCR amplification using the handout. 1.3 x 10⁷ bp in the yeast genome and thus about 6 x 10⁵ genomes in 1 µg of yeast genomic DNA. You need to devise 3 PCR experiments where we use 1.5, 1.75 and 2.0 mM of MgCl₂. The concentration of MgCl₂ is very important. Use the handout to guide you towards making a 50 µL reaction for each experiment. We want to amplify our DNA in 30 cycles. Primers come as stock solutions with concentrations of 100 µM each.

Feb. 5/7: Conduct PCR, Finish Web Pages

1) Start PCR to amplify all 5 yeast IDH genes. Here are the primers we will use.
Taq PCR Master Mix by Qiagen #201443

2) Pour 2 gels that will hold all the samples plus MW markers.

3) Make 100 mL of SOC medium. Look on page 4 of the PDF file for SOC protocol. Note: Only make 10 mL of the 1M MgSO₄•7H₂O + 1 M MgCl₂•6H₂O stock solution and 10 mL of the 2M glucose solution.

3) Final touches on your IDH web pages.