Molecular Cloning Techniques Overview

Molecular cloning is used to construct recombinant ______ molecules.

The original PCR-restriction-ligation based cloning methods are still widely used for ______-scale investigations.

E. coli is typically the host organism used for maintaining ______ DNA.

A popular choice for cloning vectors when the DNA is relatively small is a ______-based cloning vector.

Cloning vectors include a selectable ______ to allow positive selection of transformants.

Molecular cloning has applications in the production of recombinant protein products such as ______.

The final application of the cloning vector can involve protein ______ and purification.

Elements necessary for propagation and maintenance are required for plasmid ______ in E. coli.

A common technique to insert a PCR product into a cloning vector is to use restriction ______ to linearise the vector.

PCR is widely used in molecular cloning to amplify the target ______ from the chosen organism.

If the target DNA is prokaryotic, genomic DNA can be purified from ______ and directly used as a template.

To create complementary DNA, RNA can be converted using a retroviral enzyme called ______ transcriptase.

In a typical PCR reaction of 30 cycles, each target sequence can theoretically generate 2^30 copies, which is over ______ billion.

DNA polymerase synthesises new complementary DNA strands in the ______ to 3' direction during the extension step.

Agarose gel electrophoresis is used to check the purity and ______ of the PCR product.

To visualise DNA in the agarose gel, a nucleic acid ______ is used.

One commonly used fluorescent dye for staining DNA is ______ Safe.

T4 DNA ligase is used to ligate the compatible sticky ends of the vector and ______ product.

The annealing temperature during PCR should not be too ______ nor too high.

Prior to ligation, the vector DNA may be treated with Antarctic ______ to prevent self-ligation.

During the primer binding step, the temperature is reduced to ______ - 68°C.

Transformation allows the plasmid to be stably maintained and ______ for further experiments.

Samples are loaded into the wells of a polyacrylamide gel under ______, and an electric field is applied across the gel to separate proteins.

Proteins can either be separated in their native state (native PAGE) or in a ______ state by using a chemical such as sodium dodecyl sulphate.

SDS is a negatively charged anionic ______ that denatures proteins by destroying their secondary, tertiary, and quaternary structure.

In SDS-PAGE, the samples are heated to ______ °C for 5 – 10 min in a loading buffer containing SDS.

The reducing agent disrupts ______ bonds, helping to fully denature the proteins in the samples.

Coomassie Brilliant Blue R-250 is a negatively charged dye commonly used to ______ proteins in a gel following PAGE.

After PAGE, the gel is placed onto a ______ membrane, which has a high affinity for proteins.

The membrane is soaked for 1 – 2 hr in Tris-buffered saline containing 0.2% ______ to prevent non-specific binding of detection antibodies.

The primary antibody specifically binds to your ______ of interest during the Western blotting process.

The enzyme that generates smaller fragments which stimulate growth of the gland is called ______ Secretory Protease (AsP).

Disruption of pituitary function can result in rapid atrophy of the ______.

The adrenal cortex produces steroid hormones including ______ and cortisol.

AsP is produced as a zymogen, which is an ______ precursor.

The activation peptide must be cleaved before the enzyme is ______.

As a research scientist, you wish to investigate the substrate ______ and the enzyme kinetics of AsP.

To achieve both aims you need a source of pure ______

You decide to generate recombinant protein by cloning the AsP ______ into a bacterial expression vector.

His tags allow rapid purification of the protein by affinity chromatography on an immobilized ______ column.

The vector pET32A enables high expression levels in ______.

You will clone the rat adrenal secretory serine protease (AsP) cDNA into the pET32A vector to express a C-terminal ______ fusion protein.

Digestion of pET32a with NdeI and ______ will result in the release of two fragments.

Since AsP is a secreted protein, you need to create a construct that does not encode the secretory ______ sequence.

You need to include an AseI site in the forward primer and a ______ site in the reverse primer sequences.

The STOP codon is removed because you use the STOP codon present on the plasmid after the ______ tag coding sequence.

You will need to check that the AsP cDNA is correctly expressed as a recombinant C-terminal HIS tag ______ protein.

You will analyze the proteins using SDS-PAGE and ______ staining.

Following IPTG induction, you should see a band at approximately ______ kDa in the induced culture.

You check the plasmid correctness by screening transformants using colony ______.

The GeneRuler 1kb DNA Ladder mix contains ______ DNA fragments.

The Tris-acetate-EDTA running buffer consists of 0.04 M Tris-acetate; 0.001 M ______.

E.coli is not naturally competent, but there are two main methods used to transform E.coli in the laboratory by artificially inducing a temporary state of ______.

In chemical transformation, E.coli cells are treated with divalent cations, typically Ca2+ in the form of calcium ______.

During chemical transformation, E.coli cells are subjected to a temporary heat shock at typically ______°C for 30 sec.

Electroporation punctures tiny holes in the cell membrane using a temporary ______ field.

Cells that do not take up a plasmid with a selectable marker cannot survive on selective ______.

When an E.coli cell takes up a plasmid, the plasmid replicates within the cell ______.

Bacteria that have successfully taken up exogenous DNA are referred to as ______.

Colony PCR can be used to screen transformants for the presence or absence of a foreign DNA ______.

It is possible to use colony PCR to check the ______ of the insert within the MCS.

Plasmid DNA can be purified from a sample of each culture using ______ kits.

If you have successfully cloned a gene of interest into an expression ______, you can transfer your construct into a host strain.

The presence of the inducer Isopropyl β-D-1-thiogalactopyranoside (IPTG) enables T7 polymerase to transcribe the recombinant ______.

A common method to detect recombinant proteins is through ______, which separates proteins based on their electrophoretic mobility.

Acrylamide is highly toxic to humans in powder form, so pre-cast ______ gels are often used for safety reasons.