BRED: Bacteriophage Recombineering Techniques

<p>Kanamycin. (B)</p> Signup and view all the answers

<p>0.4. (A)</p> Signup and view all the answers

<p>30 minutes to 2 hours. (B)</p> Signup and view all the answers

<p>10%. (A)</p> Signup and view all the answers

<p>To isolate the plasmid for transformation. (C)</p> Signup and view all the answers

<p>100-400 ng (A)</p> Signup and view all the answers

<p>100 bp on either end (C)</p> Signup and view all the answers

<p>19-22 ms (C)</p> Signup and view all the answers

<p>To ensure successful phage mutation (D)</p> Signup and view all the answers

<p>It reduces the time constant (B)</p> Signup and view all the answers

<p>In sterile dH2O (B)</p> Signup and view all the answers

<p>It adversely affects time constants (A)</p> Signup and view all the answers

<p>50-150 ng (D)</p> Signup and view all the answers

<blockquote> <p>350 bp (C)</p> </blockquote> Signup and view all the answers

<p>To prepare for dilution to the working concentration (B)</p> Signup and view all the answers

<p>Identification of point mutations (A)</p> Signup and view all the answers

<p>Restriction enzymes (D)</p> Signup and view all the answers

<p>1:50 dilution with dH2O (B)</p> Signup and view all the answers

<p>To bind to the template DNA (C)</p> Signup and view all the answers

<p>It amplifies sequence changes around a mutation (B)</p> Signup and view all the answers

<p>At -20°C (C)</p> Signup and view all the answers

<p>To generate mutations in mycobacteriophages (D)</p> Signup and view all the answers

<p>A double-stranded DNA substrate with 100 bp flanking regions (A)</p> Signup and view all the answers

<p>It expresses recombineering functions upon induction (A)</p> Signup and view all the answers

<p>Point mutations (B)</p> Signup and view all the answers

<p>100 bp of sequence homologous on each end (A)</p> Signup and view all the answers

<p>Translocations (B)</p> Signup and view all the answers

<p>They provide a higher mutation rate (D)</p> Signup and view all the answers

<p>Performing PCR to confirm the presence of mutations (C)</p> Signup and view all the answers

<p>Decrease the annealing temperature to 2°C lower than the lowest melting temperature. (C)</p> Signup and view all the answers

<p>Using QIAquick PCR Purification Kit or MinElute Reaction Clean-up Kit. (D)</p> Signup and view all the answers

<p>100 ng/µl or greater. (D)</p> Signup and view all the answers

<p>Running approximately 5-10 µl of each reaction on an agarose gel. (B)</p> Signup and view all the answers

<p>Perform gel extraction for purification. (D)</p> Signup and view all the answers

<p>To resuspend the Extender primers to 10 µM. (B)</p> Signup and view all the answers

<p>~50-100 ng. (D)</p> Signup and view all the answers

<p>Resuspend at 1 µg/µl and dilute to 200 ng/µl. (D)</p> Signup and view all the answers

<p>To provide in-frame deletion with proper homology (D)</p> Signup and view all the answers

<p>Agarose gel (B)</p> Signup and view all the answers

<p>40-45 nt on either side of the insertion (C)</p> Signup and view all the answers

<p>To provide additional homology for recombineering (C)</p> Signup and view all the answers

<p>Phage buffer (D)</p> Signup and view all the answers

<p>2.5 mM each dNTP (B)</p> Signup and view all the answers

<p>Chloramphenicol (CB) (D)</p> Signup and view all the answers

<p>Add CaCl2 from a sterile 0.1 M stock (C)</p> Signup and view all the answers

Bacteriophage Recombineering with Electroporated DNA, a method to create mutations in mycobacteriophages.

A strain used in BRED that has high recombination efficiency due to phage proteins.

A 200 bp DNA structure used in BRED for mutations, needing 100 bp homology regions.

A mutant created by introducing a sequence flanked by homologous DNA regions.

A linear DNA structure used in BRED for gene replacement, with homologous ends.

Changes made to specific locations in the DNA sequence using synthetic oligonucleotides.

A bacterial strain containing plasmids that allow efficient DNA recombination during BRED.

A method to identify mutated plaques by amplifying specific DNA sequences after transformation.

Thermocycling involves denaturation, annealing, and extension.

Temperature where primers bind to DNA; adjust based on melting temperature.

Tool to clean-up PCR reactions, concentrating the product.

Method for checking PCR yield by separating DNA fragments visually.

The amount of DNA produced from PCR reactions, checked by gel.

The starting DNA used in PCR to replicate desired sequences.

PCR-based technique to replace specific DNA sequences using primers.

Short DNA sequences used in genetic engineering or PCR.

Sequence similarity between DNA fragments used in recombineering.

Double-stranded DNA used as a template in genetic modifications.

Primers designed to extend and provide flanking homology for PCR.

Bacterial cells made competent for transformation via electroporation.

Includes oligos, polymerase, buffer, and dNTPs for DNA amplification.

Process of introducing foreign DNA into bacterial cells.

Solution containing salts and Tris for stabilizing DNA during experiments.

Primers that anneal upstream and downstream of a mutation in the genome.

The mutant product should be distinguishable in size from the wild-type product.

Deletion Amplification Detection Assay for detecting mutations.

Mismatch Amplification Mutation Assay for detecting point mutations.

Resuspending primers in TE buffer before dilution.

The process of setting up PCR reactions with specific components.

A combined mix containing enough components for multiple PCR reactions.

A device used to amplify DNA via PCR by cycling temperature.

A linear piece of DNA used in recombineering processes.

Achieved using 100-400 ng of recombineering substrate per transformation.

100 bp of homology is sufficient for insertions, deletions, or mutations.

Cells that are capable of taking up foreign DNA during transformation.

The efficiency of transformation measured by number of colonies formed.

The ideal duration for effective DNA transformation, typically around 19-22 ms.

Testing competency with phage DNA to validate transformation success.

Residual salts in DNA can adversely affect transformation time constants.

Short synthetic DNA used to delete specific genomic regions.

A plasmid used for genetic manipulation in E. coli and M. smegmatis.

A marker used to select for transformed bacteria.

A nutrient-rich medium used to grow Mycobacterium species.

Optical Density measured at 600 nm, indicating bacterial growth.