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Unformatted text preview: Page 1 of 7 Bioengineering 115: Cell Biology for Engineers Laboratory 8b: RT-PCR Last updated: January 21, 2011 Lab 8b: RT-PCR 1 Introduction Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) is a fairly recent but generally powerful technique for biological research. While it does not lend itself to easy quantification, it can show when mRNAs for a particular protein are being made or not. This gives us a ‘snapshot’ of the cell’s genetic machinery at a particular time, and allows us to gather information regarding transcription. We have already explored experiments that deal with translation (protein electrophoresis and Western blotting). In RT-PCR, mRNA is first used to create corresponding cDNA (hence, the use of reverse transcriptase). Then, the cDNA of interest is exclusively expanded using PCR. You will qualitatively compare the expression of a gene under two conditions which will be discussed in lab. You will have lysed cells and purified RNA from one of the conditions, and you will work with another group to obtain a sample of RNA from the other condition. 2 Objectives • To create cDNA corresponding to the mRNA in your RNA sample • To amplify a portion of a particular cDNA, indicating gene expression • To compare the expression of that particular gene under two different conditions 3 References • PCR animation (http://www.dnalc.org/resources/animations/pcr.html) • RT-PCR animation (http://www.bio.davidson.edu/courses/immunology/Flash/RT_PCR.html) • Methods in Molecular Biology o History of PCR (https://docs.google.com/leaf?id=0B_YYAsAEXdBxZGFlZmFmMzctZjBhMC0 0Y2Y0LWJhOWQtZDA1NmJhMGUyZGFm&sort=name&layout=list&num=50 o Optimization of PCR https://docs.google.com/leaf?id=0B_YYAsAEXdBxYzRlNzA5MjEtM2M0NC0 0MGM2LTllMWYtNDliY2E0NjhhNzU2&sort=name&layout=list&num=50 o PCR Quality Control (https://docs.google.com/leaf?id=0B_YYAsAEXdBxYjcwY2FmMGEtOTliMC0 0N2ZkLWI4MzgtYzk4NGRmMjEzMmM1&sort=name&layout=list&num=50) o PCR Primer Design (https://docs.google.com/leaf?id=0B_YYAsAEXdBxMWZlOGRhMzgtNjg5NC 00ZGFhLWIwNjYtNzVhOWEwYWQ0YWQ0&sort=name&layout=list&num=5 0) • BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) 4 Reagents, Supplies, and Equipment Page 2 of 7 Bioengineering 115: Cell Biology for Engineers Laboratory 8b: RT-PCR Last updated: January 21, 2011 4.1 Reagents 1. Autoclaved, distilled water 2. Taq polymerase (5 units/μL solution) 3. Primers (for gene of interest, 10 μM solutions in RNase-free TE buffer) 4.2 Supplies 1. Superscript RT-PCR kit (Invitrogen 11904-018) 2. Filter-tip pipette tips 3. Thin-walled PCR tubes 4.3 Equipment 1. Mini desktop centrifuge 2....
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This note was uploaded on 10/14/2011 for the course BIOE 115 taught by Professor Haley during the Fall '11 term at City College of San Francisco.
- Fall '11
- Tissue Engineering