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Functional Genomics Research Stream
Lecture: April 7, 2009Topics: PCR, Enzyme Mediated Molecular Reactions
Agenda
• Enzymes
• Enzyme Mediated Reactions
• PCR
• Assignment Nine Progress
Enzymes
• Define?
• Examples?
• Typical Naming Scheme?
Enzymes
• Define:Molecules that catalyze the rate of some reaction. Typically proteins.
• Examples:RNase, DNase, reverse transcriptase, endonuclease, Eco-RI (restriction enzyme)
• Naming Scheme: _________ ase
Enzyme Rates
Current Protocols Essential Laboratory Techniques
Enzymes: Temperature
Current Protocols Essential Laboratory Techniques
Enzymes: pH
Current Protocols Essential Laboratory Techniques
Enzymes Assist
(process)
raw materials product
RNA polymerase II
genomic DNA ?
(transcription)
enzyme
Name This Cellular Process
Enzymes?Wikipedia
PCR
• Polymerase Chain Reaction
• Enzyme Mediated Reaction
• Typical Raw Materials: _______ ?
• Typical Products: ________ ?
DNA Representation
5’
convenientrepresentation
chemicalrepresentation
3’ 5’3’
5’
3’ 5’
3’start with double stranded DNA
5’
3’ 5’
3’denaturation
5’
3’ 5’
3’annealing of primers
5’
3’ 5’
3’
elongation by polymerase (first cycle complete)
3’ 5’
5’ 3’
second cycle complete
5’ 3’
3’ 5’
5’ 3’
3’ 5’
5’ 3’
3’ 5’
5’ 3’
3’ 5’
Current Protocols Essential Laboratory Techniques
Protocol Overview
Current Protocols Essential Laboratory Techniques
Gel Results
PCR & Assignment Nine
• You are working with a deletion strain.(a strain with a gene deleted)
• Is there some way we can actually verify the strain is lacking the deletion?
A Gene is Deleted
Gene X
kanMX4
is replaced with
Gene X
by homologous recombination
chromosome
chromosome
PCR Verification of Deletion is Possible
kanMX4
Gene Xchromosome
chromosome
pA pDpB pC
pKanBpA pD
PCR products reverse primerforward primer
pKanC
assignment nine terms
reverse primerforward primer final report
why is a positive control reaction needed?
Question: Could PCR be used to guarantee you are working with only your deletion strain?
Work This Week
Continued:Cleaning & Safety
• Reagent station: better, not great.
• Glassware: better, not great.
• 10 cultures thrown out on Monday morning.
• 10 plates need to be cleaned out of incubator now (refrigerator or trash).
• ~10 YPD bottles have been contaminatedwhich is about 9 more than I have ever seen
Contamination?
• 1 Billion Point Award*
• How is it occurring?
* no, not really
Secondary Question:What effect is contamination likely having on some of your experiments?
Solution Making Reminder
• Make Stocks!1M EDTA, 1M Tris, 1M NaCl, etc
• Dilute from Stocks!100 mM TrisTE (100 mM Tris, 10 mM EDTA)
• Reduce workload, contamination, sadness.
One Week Left(Assignment Nine)
• Nearly everyone a little (or lot) behind on progress.
• Design:Week 1 - Growth & ReagentsWeek 2 - DNA, RNAWeek 3 - PCR
• Reality:Week 1 - ?Week 2 - Growth & ReagentsWeek 3 - DNA, RNA, PCR (yowza)
A9 Completion Options
• Complete full assignment for:plus 10 pointsuse results for final report
• Complete all but Section G:minus 10 points &results still needed for final report
Final Report
• Details coming soon.
• Final assignment.
• Will be due end of semester.sometime between May 5 and May 8
• Will involve: writing, researching, experiments, results, conclusions.
Questions?
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