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Practice problems on Homologous Recombination (PDF)



Laboratory Techniques (PDF)


The following techniques are described over the course of the semester in 7.28.


LEC #TOPICSTECHNIQUESASSAYS FOR
1Replication (Replication Enzymes)dNTP Incorporation AssayDNA Synthesis (Fast; Quantitative for Amount of Synth.).
Filter Binding AssayMethod to separate incorporated from unincorporated dNTPs.
Gel ElectrophoresisMethod to separate DNA on the basis of length.
Primer Extension AssayDNA Synthesis (Slow; Quantitative for Length of Synth.).
Template Challenge AssayAssay for Processivity.
2Replication (Replication Fork)DNA Helicase AssayIn vitro DNA Helicase Activity.
Helicase Polarity AssayAssay for directionality of helicase movement; used to study function of replication termination sequences.
Topoisomerase AssayAssay for changes in DNA topology; distinguish supercoiled, relaxed, nicked, linear, forms of DNA and measure catenation/decatenation.
3Replication (Prok. System)2-D Agarose GelsAssay for Origin of Replication (Must have an idea of where the origin is to test).
Southern BlottingIdentifies specific DNA molecules transferred to membrane.
DNA Microarray AssayAssays the relative amount of replicated DNA sequences during a synchronized round of DNA replication to identify sites of replication initiation.
Plasmid Replicator AssayIdentifies region of DNA sufficient for Replicator activity.
Mutational Mapping AssayIdentifies regions of DNA necessary for Replicator activity.
Biochemical FractionationMechanism to purify biochemical activity that can be assayed in vitro.
Biochemical ComplementationCombines mutant extracts with biochemical fractionation to identify proteins required for assay (in this case DNA replication).
4Replication (Euk. Systems)Genetic Screen for DNA Replication Mutants
DNase I Protection AssayAssay for Sequence Specific DNA Binding (Slower and not quantitative; Gives Information about bound DNA sequence).
Gel Mobility Shift AssayAssay for Sequence Specific DNA Binding (Fast and Quantitative but less information about bound sequence).
DNA Unwinding AssayAssays for formation of ssDNA through the use of a ssDNA specific nuclease.
5Chromatin AssemblyTemplate Association AssayUses gel filtration to separate molecules bound to a plasmid from molecules that are not bound to a plasmid.
6DNA Mismatch RepairHeteroduplex DNA analysisAssay for DNA repair in vivo.
Restriction AnalysisAssay for methylation state of DNA.
Restriction site repairAssay of DNA repair in vitro; also used incorporation assay.
7DNA Repair IIAmes TestAssay for chemicals and other factors that increase the frequency of mutations.
Nicking of Circular DNAAssay for excision-type mechanism of DNA repair.
Lesion bypass DNA synthesisAssay for ability of replication/repair proteins to synthesize DNA off of a damaged template.
8DS Break Repair, Homologous RecombinationStrand Exchange AssayAssay for homologous DNA pairing and branch migration.
9Homologous Recombination EnzymesATPγSUsed in determining whether ATP binding or ATP-hydrolysis is required for a reaction.
DMS FootprintingAssay for protection of the bases in DNA, rather than the phosphodiester backbone.
Cooperative DNA BindingGel-shift assay to measure cooperative vs. non-cooperative DNA binding.
10Meiotic RecombinationConsensus SequencesIdentification of important elements by conservation.
Native-Denaturing 2D GelDetection of nicks vs DSBs in DNA.
11Site-specific RecombinationDNA Bending AssayAssay for whether a protein bends DNA upon binding and position of bend.
12TranspositionGenomic SouthernIdentifies specific DNA molecules transferred to membrane (here used for transposition).
Plasmid MatingAssay for transposition in vivo by determining movement of drug resistance to a new plasmid.
13Transcription: Prok. Machinery and PromotersIncorporation AssayAssay for Transcription in vitro.
S1 Protection AssayAssay for Transcription. Maps start site of transcription.
Northern BlotAssay for Transcription. Maps steady state RNA in vivo.
Microarray Analysis of RNAAssay for relative levels of gene expression. Can assay many genes at once.
Pulse Labeling of in vivo RNAAssay for actively transcribing genes.
Promoter Fusion AssayAssay to indirectly measure promoter activity.
14Transcription: Euk. Machinery and PromotersDNA Unwinding AssayAssay for open complex formation.
Indirect end-labelingMethod to extend a labeled DNA primer to measure breaks or modification of DNA.
15Transcription: Regulation IAntibody Supershift AssayAssay to determine the components of protein-DNA complex in a gel shift assay.
16Transcription: Regulation IITranscription Factor DNA Binding Domain Mapping
Transcriptional Factor Activation Domain Mapping 
Chromatin ImmunoprecipitationAssay to determine the in vivo binding site of proteins.
17RNA Processing IR-Loop FormationAssay for regions of mRNA that anneal to different DNA fragments.
Gel Electrophoresis AssayFor intermediates and products formed during splicing in vitro (done using labeled RNA).
18RNA processing IICalculation of "Consensus Fold" and Rnase DigestionDetermination of RNA secondary structures.
19RNA Processing: RegulationcDNA CloningDetermine mRNA sequence and structure, protein expression.
RT-PCRGenerate segments of amplified DNA starting with specific RNA template.
Exon TrappingFind exons in DNA fragments of regions thought to contain genes.
20RNA Editing, RNAi and miRNAs
21Translation: Basic Machinery I  
22Translation: Basic Machinery II
23Translation: Regulation IIncorporation Assay for Translation Polysome AssayIdentifies mRNAs that are being translated in vivo.
Gel Filtration Analysis of Protein BindingMeasures binding of small molecules to larger molecules (e.g., binding to ribosome).
Filter Binding AssayMeasures binding of RNA to protein.

 








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