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 MgSO47H2O + 1 M MgCl26H2O stock solution and 10 mL of the 2M glucose solution.
3) Final touches on your IDH web pages.
Feb 12/14: Clean PCR, verify on gel, ligate, transform into expression cells
1) Clean PCR products. Elute in 30
QiaQuick PCR Purification Kit #28104
2) OD purified PCR product with 40 fold dilution.
3) Calculate how much PCR product to get 10X excess or 4 µL, which ever is less.
4) Ligate PCR
products into expression vector. Ligate for 1 hour.
QiaExpress UA Cloning KIt #32179
Vector may need SAP.
5) Transform ligation into JM109 strain
of E. coli.
Feb. 19/21: Screen for insert and orientation
1) Night before, pick colonies and grow them overnight (O/N)
2) Minprep plasmid DNA
QiaPrep Spin MiniPrep #27104
4) Verify identities and orientations
of PCR products with restriction digestions. Electrophorese
Download copy of 1kb ladder PDF.
Feb. 26/28: Southern Blot part 1
1) Continue to hunt for IDH genes in forward orientaitons
March 12/14: Southern Blot part 2
1) Run gels and photograph them with ruler
2) Transfer Southern Blot
3) Make probes
March 19/21: Express IDH Proteins
1) Day before lab, add probes to blots.
2) Finish Southern Blot
March 26/28: Run Western Blot part 1
1) Night before lab, begin O/N cultures of forward and reverse clones.
2) Induce protein production.
3) Affinity purify IDH proteins.
Qiagen Ni-NTA Spin Kit #31314
4) Freeze in aliquots.
April 9/11: Finish Western Blot
1) Run SDS-PAGE.
2) Transfer to membrane.
April 16/18: Run IDH Enzyme Assays
1) Bind antibody and detect prtoeins.
2) Determine MW of all 5 IDH proteins.
April 23/25: Final Wrap Up
1) Verify NAD+ and NADP+ specificity.
May 8 at noon: Turn in Lab Notebooks and Lab Reports -
1) Your lab notebooks are due at noon May 8.
2) Your lab reports are due at noon May 8.
Lab Schedule In Context of Research Project
Molecular Biology Main Page
Biology Main Page