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?