GN701 molecular methods

how to convince bacteria to replicate transformed genes (put genes in plasmid and make bacteria take it up now how do you make it make more of them)

"make" this plasmid essential for the survival of bacteria have the plasmid code for antibiotic resistance then grow on antibiotic plate

Each ZF motif recognizes

3 specific nucleotides in the target DNA

DNA sequencing: the Maxam-Gilbert method

5'-ends of dsDNA are radioactively labeled by polynucleotide kinase and [g-32P]-ATP dsDNA is denatured in the presence of DMSO and heat, and ssDNA is purified* Labeled ssDNA is split into 4 reactions, chemically treated and cleaved after specific bases Obtained fragments are separated on a polyacrilamide gel and autoradiographed**

DNA Subcloning

A combination of restriction enzymes and DNA ligase can be used to generate recombinant DNA molecules in a plasmid/vector by means of subcloning Typically, one or multiple pieces of foreign DNA (e.g. a gene from humans) is inserted into a bacterial plasmid vector (e.g. pUC18) and transformed into bacteria Bacteria will replicate the insert-harboring plasmid and produce millions of its copies

Comparison between normal dTTP and "terminator" ddTTP Sanger sequencing

A dideoxynucleotide (for example, dideoxythymidine triphosphate, ddTTP) lacks 3' hydroxyl group, 3′-OH Lack of 3′-OH prevents the next nucleotide from attaching => DNA synthesis terminates

Nanostrings

A hybridization-based method that employs two gene-specific probes per gene, a capture probe B and a reporter probe A (the CodeSet) Both probes have a region of homology (35-50 bases each) to the gene (or transcript) of interest (GOI) and will thus specifically bind to that gene A reporter probe also carries a unique multi-color fluorescence tag that serves as a "bar-code" or "identifier" for the GOI the probe recognizes A capture probe is tagged with biotin that enables purification/immobilization of the GOI recognized by the probe using streptavidin Up to 800 probe pairs can be used in a single hybridization reaction, thus enabling detection and quantification of 800 genes or transcripts at once in a complex sample (e.g., a total RNA prep from a tissue of interest) After the hybridization step (in this case, between mRNA and the CodeSet), probe-bound transcripts are purified and electrophretically aligned on a streptavidin-coated nCounter cartridge The fluorescent barcodes are imaged and tallied

Pacific Biosciences Single Molecule Real Time (SMRT)

A single DNA polymerase enzyme is affixed at the bottom of a zero-mode waveguide (ZMW) well with a single molecule of DNA as a template. The ZMW is a structure that creates an illuminated observation volume that is small enough to observe only a single nucleotide of DNA being incorporated by DNA polymerase. Each of the four DNA bases is attached to one of four different fluorescent dyes. When a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is cleaved off and diffuses out of the observation area of the ZMW where its fluorescence is no longer observable. A detector detects the fluorescent signal of the nucleotide incorporation, and the base call is made according to the corresponding fluorescence of the dye.

Oxford Nanopore (MinION)

A single strand of DNA is threaded through a tiny protein pore in a synthetic membrane An electric current flows through the pore Different DNA bases disrupt the current in different ways The machine measures the current and interprets the sequence Drawbacks: higher error rates and lower throughput Pluses: portability and ease of use; can also sequence RNA

Bacterial plasmid DNA extraction

Alkaline lysis protocol: -Grow bacteria o/n -Collect by centrifugation -Resuspend in GTE -Lyse in NaOH/SDS -Neutralize with KoAC -Spin to remove debris -Isolate DNA from s/n by: -Alcohol precipitation, or -Membrane purification

The origin of replication

An AT-rich region of DNA where replication starts Determines plasmid copy number by controlling how often the plasmid replicates Plasmid replication always relies on the host replication machinery but can be independent of chromosomal DNA replication and host cell cycle

Immunolocalization

An antibody-based detection method for proteins and other biomolecules directly in a cell, tissue, or fractionated lysate Relies on similar principles (i.e. antigen-antibody interactions) as Western blot An antibody is tagged with a fluorophore, an enzyme, or gold particles to enable antigen detection

SDS-PAGE

An electrophoresis technique that separates proteins by size Tissue samples are treated with a reducing agent (dithiothreitol or beta-mercaptoethanol) to break disulfide bridges in proteins and with the detergent sodium dodecyl sulfate (SDS) to solubilize proteins and coat them with a negative charge Samples are boiled (to denature the proteins) and subjected to polyacrylamide gel electrophoresis (PAGE) SDS-coated denatured proteins get separated by molecular weight: smaller proteins travel faster larger proteins travel slower

BiFC

Bimolecular fluorescence complementation or split YFP

ways to transform bacteria (make accept extraneous DNA)

Chemically competent cells are prepared by re-suspending cells in CaCl2 solution (which permeabilizes E. coli cell membranes) and subjecting the cells to a mild heat shock (~42oC) Electrocompetent cells are prepared by washing the cells with water to remove salts and subjecting the cells to a short electric pulse

Insert orientation testing by restriction digest or pcr

Choose an enzyme (or a pair of enzymes) that cuts in the vector and asymmetrically in the insert Set up a PCR reaction using a forward vector primer plus a reverse insert-specific primer (or a forward insert-specific primer plus a reverse vector primer) Primers will only amplify if the insert is present and is in the right orientation

ChIP

Chromatin immunoprecipitation

cDNA

Complementary DNA. DNA produced synthetically by reverse trascribing mRNA. Because of eukaryotic mRNA splicing, cDNA contains no inrons.

DNA manipulation techniques:

DNA extraction/purification Restriction digest DNA ligation (as part of subcloning) Transformation Gel electrophoresis Polymerase chain reaction (PCR) DNA sequencing

Strategies that complement next-gen sequencing

DNA microarrays NanoString qPCR Optical mapping

DNA sequencing: Sanger chain termination (dideoxy) method

DNA sequencing reaction is an in vitro DNA replication reaction that proceeds until DNA polymerase incorporates a fluorescently labeled dideoxynucleotide "terminator"

Restriction Endonucleases

Different restriction enzymes make different types of cuts and produce (A) 5' overhangs, (B) blunt ends, or (C) 3' overhangs Restriction enzymes bind to dsDNA and cut phosphodiester bonds in both strands of the DNA double helix

ddPCR (acronym for)

Droplet digital PCR

Electrophoretic mobility shift assay (EMSA)

EMSA is a common electrophoretic technique for assaying protein-DNA interactions in vitro Radioactively labeled DNA fragments of interest are incubated with the protein of choice, run through a polyacrylamide gel, and the gel is exposed to film If the protein is bound to the DNA, the DNA band migrates slower through the gel and appears shifted on an autoradiogram

EMSA

Electrophoretic mobility shift assay

Reporter genes: GFP

Encodes a 238 aa protein that fluoresces bright green color under UV light (excitation at 395 nm and emission at 509 nm)

Ethidium Bromide

Ethidium Bromide EtBr is an intercalating agent that inserts itself between the stacked base pairs of DNA EtBr-bound DNA glows under UV light, making it possible to visualize DNA fragments on a gel

SOLiD sequencing by ligation how many rounds likely done?

Five rounds of primer reset every base is interrogated in two independent ligation reactions by two different primers

FRET

Fluorescence (or Förster) resonance energy transfer

How do GFP reporters work? What did you mean by saying "functionality of a reporter needs to be confirmed by complementation"?

GFP fluorescence highlights the tissues/cells where the gene of interest is expressed: With translational fusions GFP also gives you another level of information, i.e. where the protein of interest is

How does GFP know which cells/tissues are expressing the gene of interest? Is GFP gene specific?

GFP is not a fluorescent dye, but a protein encoded by a gene. it is expressed from the promoter of a gene of interest (and hence is expressed only in the cells that normally express the gene of interest). GFP is not gene-specific it can be fused (in a DNA construct) to the gene of interest in frame with your gene to preserve your gene activity. This is what enables it to "report" on the localization of the fusion protein, not some sort of sequence-specific binding of GFP to the gene or protein of interest.

Reporter genes: GUS and LacZ

GUS and LacZ encode enzymes, beta-glucuronidase and beta-galactosidase, that convert colorless substrates (X-gluc and X-gal) to blue products The substrate can be infiltrated into the transgenic tissues to visualize where GUS or LacZ are expressed

Gene expression analysis techniques:

Gene expression analysis techniques: Reporter genes Transcriptional and translational reporters Northern blot In situ hybridization Reverse transcription-PCR (RT-PCR), qRT-PCR and ddPCR Nanostrings RNase protection assay Microarrays (cDNA and oligo arrays) Next-gen sequencing (RNA-Seq)

Which primer(s) would you use for RT-PCR to amplify the transcript of interest by PCR?

Gene-specific, i.e. different primer pairs for each gene; PCRs on several genes are typically done on the same cDNA samples to enable direct comparisons of gene expression patterns

Single-molecule or 3rd generation sequencing (~2014)

Helicos Biosciences Pacific Biosciences Single Molecule Real Time (SMRT) Oxford Nanopore

DNA-DNA or DNA-RNA hybridization

Hybridization is based on sequence complementarity between the target molecule and the gene-specific probe

Droplet digital PCR (ddPCR)

In ddPCR, a PCR sample is emulsified in oil to produce ~20,000 droplets. The resulting distribution of template molecules among the droplets is random, so that some droplets have no template molecules and others have one or more. Each droplet corresponds to a separate PCR amplification and is analyzed separately The droplets are then individually counted and scored as positive or negative for fluorescence to infer the absolute number of template molecules in the starting sample using Poisson Statistics

Yeast two hybrid (Y2H)

In vivo technique Y2H is based on the modular architecture of transcription factors, that contain a DNA-binding domain (DBD) and a transcription-activation domain (AD) DBD recognizes and binds to a particular promoter element, whereas AD interacts with the RNA polymerase II complex and activates transcription DBD and AD can be expressed in yeast as separate peptides and brought physically together by an interacting pair of "bridge" proteins, the bait and the prey Neither DBD fused to the bait nor the AD fused to the prey can activate transcription of the reporter gene on their own But if the two fusions are co-expressed in the same yeast cell and the bait and the prey physically interact, then DBD and AD are brought together and are able to activate transcription of the reporter gene

RNA immunoprecipitation (RIP)

Is similar to protein co-IP, except that the downstream readout of RNA is by RT/qRT-PCR (targeted approach) or RNA-Seq (non-targeted approach) CLIP (cross-linking immunoprecipitation) is RIP that involves UV crosslinking of RNA/protein complexes prior to immunoprecipitation

Reporter genes: Luciferase

Luciferase encodes an enzyme that catalyzes oxidation of a pigment luciferin in a bioluminescent reaction that emits light

Classical or 1st generation sequencing (1977)

Maxam-Gilbert chemical modification and cleavage Sanger dideoxy chain-termination

Bacterial plasmid: pUC18/19 vector

Multiple Cloning Site in a plasmid contains recognition sequences for several different restriction enzymes

Gel electrophoresis

Negatively charged DNA travels through the gel towards the positive pole in the electrophoresis chamber Smaller DNA fragments travel faster than larger fragments, allowing separation of the fragments based on size (and shape!)

Possible tests to confirm the identity of a recombinant plasmid

Perform a restriction digest on a the DNA from the white colony and run the products on a gel (restriction digest + gel electrophoresis) Run a PCR reaction using gene-specific primers Sequence the insert and its junction with the vector

454 Pyrosequencing

Pooled amplicons are clonally amplified in droplet emulsions. Isolated DNA-carrying beads are loaded into individual wells on a PicoTiter™ plate and surrounded by enzyme beads. Nucleotides are flowed one at a time over the plate and template-dependent incorporation releases pyrophosphate, which is converted to light through an enzymatic process. The light signals, which are proportional to the number of incorporated nucleotides in a given flow, are represented in flowgrams that are analyzed and a nucleotide sequence is determined for each read with the GS Amplicon Variant Analyzer software. This is a sequencing-by-synthesis approach that using reactions to detect pyrophosphate (PPi) released during base incorporation

RPA (acronym for)

RNase Protection Assay

RNase Protection Assay (RPA)

RPA is a sensitive method for detecting and quantifying specific mRNA transcripts in a heterologous population of RNAs (e.g. total RNA from a tissue) Total RNA is hybridized to a radioactively labeled antisense probe (RNA or DNA) complementary to the mRNA of interest to make double-stranded RNA or RNA-DNA hybrid The mixture is then treated with RNases to digest single-stranded RNA, leaving the "protected" dsRNA or RNA-DNA hybrids intact RNase-treated samples are then separated on a polyacrylamide gel and visualized by autoradiography

reagents for pcr

Reagents needed: - DNA template - DNA polymerase - DNA primers* (F+R) - dNTPs - Buffer with Mg++

Reporter genes

Reporters are genes that are used in molecular biology to monitor expression of genes of interest (GOI) Reporters are fused to promoters, gene fragments or entire GOI and their activity is assayed/analyzed Most commonly used reporters are green fluorescent protein (GFP) and its various derivatives (YFP, CFP, etc.), luciferase, LacZ or GUS

where are Reporter gene placed

Reporters can be fused to (A) the promoter of a gene of interest, or (B) to the entire gene

Next-gen or 2nd generation or massively parallel sequencing (2000s)

Roche 454 pyrosequencing Illumina (Solexa) sequencing by synthesis Life technologies (Applied Biosystems) SOLiD sequencing by ligation Life technologies Ion Torrent semiconductor

Protein analysis techniques:

SDS-PAGE Western Blot Immunolocalization Affinity purification

Quantitative real-time RT-PCR (qRT-PCR)

SYBR Green qRT-PCR takes advantage of the SYBR Green dye that binds exclusively to dsDNA and becomes fluorescent upon binding The more dsDNA is synthesized in PCR, the more dye binds, the more fluorescence is detected by a photometer, enabling sensitive quantification of the DNA template By measuring the amount of fluorescence in a sample after each PCR cycle, the amplification can be monitored in real time

RNA electrophoretic mobility shift assay (EMSA)

Same idea as DNA EMSA, except that RNA is labeled, co-incubated with a protein of interest, the complex is resolved in a polyacrylamide gel and autoradiographed

RNAse I footptinting

Same idea as DNAse I footprinting, except that a protein/end-labeled RNA mixture is treated with RNAse I, resolved on a gel and auto-radiographed

Enzymology of pyrosequencing

Sulfurylase regenerates ATP (out of APS and PPi) Luciferase hydrolyzes ATP in the presence of luciferin, producing light Apyrase inactivates extra nucleotides (not incorporated in the DNA)

In situ hybridization: the DIG method

The digoxygenin (DIG) labeling method is based on a steroid isolated from plants of the genus Digitalis, the only natural source of DIG. DIG can be synthetically linked to the C-5 position of uridine nucleotides via a spacer arm containing eleven carbon atoms: The DIG-labeled nucleotides are incorporated into gene-specific RNA probes Hybridized DIG-labeled probes are detected with antibodies conjugated to an enzyme or a fluorescent dye

SOLiD sequencing by ligation

The first few steps, including emulsion PCR on the beads, are the same as in 454, but 3' ends of the PCR fragments are modified to enable their covalent attachment to a glass slide Beads are deposited onto glass surface and the sequence of the DNA fragments they carry is determined using a set of 16 colored "interrogation probes" Each probe is an octamer, made of (3′-to-5′ direction!) 2 probe-specific bases and 6 degenerate bases (nnnzzz) with one of 4 fluorescent labels linked to the 5′ end The 2 probe-specific bases in the 3' represent one of 16 possible 2-base combinations Adapter sequence on the template-carrying beads is recognized by a specific primer 4 of 16 interrogation probes are then added at a time and compete for ligation to the sequencing primer If there is a sequence match between the template and the two 3'-terminal bases of the probe, ligase covalently links the 5' end of the primer with the 3' end of the probe => fluorescence is read All four sets of probes are tested, but only one of 16 probes matches the template and can be ligated After fluorescence has been read, the three 5' zzz nucleotides and the fluorescent label are cleaved off with AgNO3 on a phosphorothiolate bond and a new round of sequence interrogation by labeled probes is initiated Multiple cycles of ligation, detection and cleavage are performed with the number of cycles determining the eventual read length Following a series of ligation cycles, the extension product is removed and the template is reset with a primer complementary to the n-1 position for a second round of ligation cycles.

Ion Torrent semiconductor

This is also a sequencing by synthesis approach but no modified nucleotides or optical detectors are used. The wells with template-attached beads are flooded with dNTPs, one kind at a time. When the nucleotide is incorporated into the growing complementary strand, a hydrogen ion is released -> a sensor reads this pH change as an electric signal. If homopolymer repeats are present in the template, multiple dNTP molecules get incorporated in a single cycle -> a larger number of protons is released and a proportionally higher electronic signal is detected.

DNase I footprinting

This method tests for protein-DNA interactions and identifies the exact regions on the DNA fragment that are bound by proteins An end-labeled dsDNA fragment is incubated with the protein(s) of interest, subjected to DNase digestion, run on a polyacrylamide gel, and autoradiographed DNase cuts DNA except for the regions protected by bound proteins that leave "footprints" on an autoradiograph

Northern Blot Analysis

This technique is used to study expression (levels and patterns) of the genes of interest Used to determine whether gene is actively being expressed in given cell or tissue Used to study patterns of gene expression in embryonic tissues, cancer, and genetic disorders

Microarrays

Thousands of nucleic acid sequences are arranged in grids on glass or silicon. DNA or RNA probes are hybridized to the chip, and a scanner detects the relative amts of complementary binding. Used to profile gene expression levels or to detect single nucleotide polymorphisms (SNPs). Microarrays can be used to analyze expression levels of hundreds or thousands of genes at a time or to genotype multiple regions of a genome by hybridizing the chips to labeled probes

applications of RT-PCR

To generate a cDNA library of all expressed genes in a tissue (cDNAs can be subcloned into a vector and transformed into bacteria or ligated to adapters, PCR amplified and sequenced) To generate a cDNA or cRNA probe to hybridize to a microarray To amplify and sequence possible splicing variants for a gene of interest To amplify and sublcone the cDNA for the gene of interest for expressing it in yeast

Yeast One Hybrid (Y1H)

Y1H is a molecular technique aimed at identifying proteins (transcription factors/TF) that interact with the DNA sequences of interest (e.g., a promoter fragment of your favorite gene) in yeast A TF of interest (or a cDNA library) is fused to an activating domain (AD) and tested in yeast for the ability to bind to the promoter region in question (the bait) subcloned upstream of a reporter gene (again, an auxotrophic or a colorimetric marker)

Yeast three hybrid (Y3H)

Y3H tests for interaction between a protein (Y) and an RNA (X) of interest Positive interaction results in growth of yeast in His-depleted media and blue color of colonies

Bimolecular fluorescence complementation (BiFC) or split YFP

YFP (or another fluorescent protein) is split into two halves (i.e., N- and C-terminal fragments) that are fused to the two proteins of interest, i.e. A and B The two halves of YFP will be brought into close proximity, reassemble together and fluoresce if A and B interact with each other Positive YFP fluorescence = interaction; no fluorescence = no interaction

Protein-DNA interactions

Yeast one hybrid Electrophoretic mobility shift assay (EMSA) DNase I footprinting Chromatin immunoprecipitation (ChIP)

Protein-protein interactions

Yeast two hybrid Pull-down assay Co-immunoprecipitation assay Fluorescence resonance energy transfer (FRET) Bimolecular fluorescence complementation (BiFC) or split YFP

Zinc finger (ZF) nucleases

Zinc fingers (ZFs) are small protein structural motifs that can coordinate one or more zinc ions to help stabilize their folds

azidothymidine (AZT)

a drug used to treat AIDS. AZT (which is also called zidovudine) is taken up by cells where it is converted into the triphosphate. Because AZT has no 3′ -OH group, DNA synthesis by reverse transcriptase halts when AZT triphosphate is incorporated in the growing DNA strand.

Co-immunoprecipitation assay (Co-IP)

an affinity purification technique based on the interaction between an antibody and antigen Cells are lysed and the extract is incubated with the AB against the protein of interest incubated with a secondary AB coupled to a solid resin that can be centrifuged to collect the protein bound resin resin is washed to remove unbound proteins and the "precipitate" is analyzed by SDS-PAGE and Western blotting and/or Mass Spec

Fluorescence In Situ Hybridization (FISH)

commonly used to investigate chromosomal abnormalities in humans or for species identification in plants and animals cytogenetic technique for the detection and localization of specific DNA sequences on chromosomes uses fluorescent DNA probes that bind to specific chromosomal regions with high degree of sequence similarity to the probe

Alkaline phosphatase

conjugated to the anti-DIG AB) converts a chromogenic BCIP/NBT substrate to an insoluble dark-purple product, enabling visualization of the sites of expression of the mRNA complementary to the DIG-labeled probe

CLIP

cross-linking immunoprecipitation

DIG labeling method

digoxygenin (DIG) labeling method

Auxotrophic markers

enable mutant yeast strains to grow on media lacking a particular amino acid or a nitrogenous base (e.g. HIS3 and URA3 allow yeast to grow on media without histidine and uracil)

Colorimetric markers

enable visual selection of yeast colonies harboring interacting proteins (e.g. LacZ gene would make yeast cells grown on X-gal blue)

In situ hybridization

enables detection and localization of mRNAs Gene-specific probes are labeled and hybridized Probe hybridization is detected using autoradiography (A), fluorescence microscopy (B) or immunohistochemistry (C)

reporter genes used to monitor what

expression patterns of the GOI at the transcriptional (A) or translational (B) level

TaqMan

fluorescent reporter probe (e.g. TaqMan) complementary to an internal fragment in the gene of interest The TaqMan probe is tagged with a fluorescent dye reporter (R) at the 5'-end and a quencher (Q) at the 3'-end

Yeast two hybrid (Y2H) used to test

for protein-protein interactions in yeast

Helicos - true single molecule seqencing

glass surface covered multi "T" ssDNA capture ssDNA to be seq, with "A" end so orientated correctly one molecule per chip add fluoro tagged nTP get fluoro image after each incorporation alternate bp and build dsDNA molecule from bottom up

Western blot also known as

immunoblot

Pull-down assay

in vitro technique In this case, the bait is bound to an affinity purification resin (e.g. a GST-tagged bait is bound to a glutathione resin, or His-tagged bait bound to a nickel resin) and a cell lysate containing the prey protein is co-incubated with the resin to allow for binding The resin is washed and then the bait bound to its prey is eluted The identity of the prey protein (if unknown) can be identified by mass spectrometry

Sequence by synthesis

ion torrent 454 illumina helicos Pacific Biosciences Single Molecule Real Time (SMRT)

Western blot

is a laboratory technique utilized to detect and quantify proteins in a biological sample After subjecting the sample to SDS-PAGE, the proteins are transferred from the gel to a membrane (nitrocellulose or PVDF) by electric current Proteins on the membrane are then detected with antibodies (AB)

Bacterial transformation

make bacteria accept extraneous DNA

Fluorescence (or Förster) resonance energy transfer (FRET)

non-radiative transfer of energy from an excited donor molecule to an acceptor molecule located in close proximity Excitation of a donor fluorophore results not only in donor emission, but also in emission characteristic of an acceptor fluorophore Two fluorophores are commonly used, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) CFP is activated by a laser at ~430nM and will emit light at ~480nm and/or transfer some of the energy to YFP that will emit at ~530nM By fusing one gene of interest to CFP and another to YFP and expressing these constructs back in the organism of choice the interaction between the two fusion proteins can be tested by FRET

RPA can be used to

quantify the relative abundance of transcripts of interest (the intensity of the "protected" band on an autoradiogram is indicative of the expression level in the tissue assayed) RPA can be employed to precisely map mRNA 5' and 3' termini and intron/exon junctions.

Which primer(s) would you use for RT-PCR to reverse transcribe mRNAs into cDNAs?

random or oligo dT, so that the same cDNA sample can be used to amplify several genes by PCR to enable comparisons between genes

The origin of replication for high copy

random partitioning occurs

The origin of replication for low copy

replication is coordinated with chromosome replication

SDS-PAGE (acronym for)

sodium dodecyl sulfate polyacrylamide gel electrophoresis

Chromatin immunoprecipitation (ChIP)

technique that tests the ability of a protein of interest to associate with particular fragments of DNA or chromatin in vivo A protein and associated chromatin in a cellular lysate are cross-linked (with UV or chemically), the DNA is sheared, and the DNA fragments associated with the protein of interest are immunoprecipitated using an AB against that protein DNA fragments co-precipitated with the protein of interest are purified and tested by PCR, sequencing, or microarrays

subcloning refers to

to cutting and ligating desired pieces of DNA together in a vector

What type of a reporter (transcriptional or translational) will you use to study: Subcellular localization of the protein of interest

translational

What type of a reporter (transcriptional or translational) will you use to study: Tissue-specific pattern of expression of a gene of interest

translational or transcriptional depending on what youre looking for

What type of a reporter (transcriptional or translational) will you use to study: Inducibility" by heat shock of the gene of interest?

translational to know what making it unstable, transcriptional to see where the gene was transcribed

DNA Ligase

type of enzymes can be used to ligate (i.e. re-attach) blunt or sticky ends

Mass spectrometry (MS)

used to infer the identity of the protein(s) in a sample by measuring the mass-to-charge ratio of ionized protein fragments

Reverse transcription-PCR (RT-PCR)

variation of PCR where the DNA template for amplification is generated by reverse transcription of mRNA mRNA of interest is reverse-transcribed into complementary DNA (cDNA) by Reverse Transcriptase cDNA can then be used as a template for PCR or quantitative real-time PCR (qRT-PCR)