Overview

Description

CALENDAR DESCRIPTION:

Modern molecular biological and recombinant nucleic acid methods will be covered. Examples are DNA and RNA isolation, plasmid preparation, restriction enzyme digestion, DNA cloning and polymerase chain reaction. Students with credit for BISC 357 may not take this course for further credit.

COURSE DETAILS:

This course will introduce students to recombinant nucleic acid methods. Lab time will involve one full afternoon every week, for which attendance is mandatory—unexcused absences will be penalized.

Laboratory Exercises:

1. Check-in, safety, Pipette practice, NanoDrop
2. Making LB Bacterial plates, casting and loading agarose gels, streaking pJ014 and pJT0 cells onto LB-Amp plates           
3. Inoculation of liquid culture from pJT0 plates, colony PCR, plasmid miniprep, PCR confirmation via gel electrophoresis    
4. Restriction digest of PCR product and purified pJT0 plasmid; gel electrophoresis; gel purification of PCR product           
5. Dephosphorylate of pJT0 vector, ligation, transformation in DH5α cells; Pour LB-Amp     
6. Site-directed mutagenesis via PCR cycling; DpnI digestion   of parental plasmid and transformation/plating       
7. Fluorescent characterization of mCherry & mutants
8. Isolation of genomic DNA and total RNA from Drosophila, NanoDrop.       
9. Formaldehyde gel analysis, PCR of gene-specific fragment from genomic DNA; generation of a specific cDNA via RT-PCR; pour LB-Amp plates
10. Gel electrophoresis of the genomic PCR product and cDNA from RT-PCR; spin column purification; cloning of the genomic PCR product into pGEM-T Easy: ligation and transformation   
11. Miniprep of pGEM-T Easy – genomic PCR, diagnostic restriction digests and gel electrophoresis of cloned product; cycle sequencing of genomic and cDNA clone         
12. Computer lab for analysis of Drosophila sequencing data

Experiments are subject to change or rearrangement.

Grading

Materials

MATERIALS + SUPPLIES:

Safety goggles or face shield, and a lab coat are required.

RECOMMENDED READING:

REQUIRED READING NOTES: