DNA & plasmid extraction
This buffer is used in the final step of the spin-column protocol to elute the purified plasmid DNA from the membrane.
Buffer EB
This specific apparatus, complete with a dark viewing cover and UV light, is used to run and visualize the agarose gels.
BlueGel electrophoresis
This is the specific, ligase-free cloning method used to insert the amplified α-amylase gene into the p15TV-L plasmid.
Ligation independent cloning (LIC)
This specific strain of E. coli is utilized as the host cell because it is optimized for recombinant protein expression.
BL21
This halogen solution is added at the end of the enzymology assay to stain undigested starch a dark blue or purple color.
Iodine
During the genomic DNA extraction of Bacillus subtilis, this enzyme is added to break down proteins in the cell
Proteinase K
According to the programmed miniPCR thermocycler protocol, this is the exact number of cycles required to amplify the α-amylase gene.
30 - 35 cycles
When transforming the chemically competent DH5α cells, the heat shock step must be performed at 42°C for exactly this duration.
30 seconds
This chemical analog of lactose is added to the bacterial culture at mid-log phase to induce the transcription of the T7 RNA polymerase and the target protein.
IPTG
This specific plant-derived carbohydrate is provided at a concentration of 2 mg/mL to serve as the substrate for the α-amylase enzyme.
Casava starch
Adding this specific neutralizing buffer causes the solution to turn colorless and precipitates the genomic DNA while leaving the plasmid soluble.
Buffer N3
To prepare the gel matrix for checking PCR and plasmid quality, you must dissolve this substance in water and melt it using a microwave.
Agarose
This specific restriction enzyme is used to digest and linearize the empty p15TV-L plasmid before the gene insertion step.
BseRI
The engineered α-amylase binds tightly to the purification column because it was cloned to contain this specific 6-amino-acid tag.
His-tag
Adding 20 µL of this strong acid denatures the enzyme and safely stops the starch hydrolysis reaction.
HCl
After the wash steps, the column must be completely dried because trace amounts of this chemical can severely inhibit downstream enzymatic reactions like PCR.
Ethanol
During the amplification of the B. subtilis α-amylase gene, the miniPCR machine’s annealing temperature is set to this specific degree Celsius.
58°C
To counter-select and kill bacteria that took up an empty, non-recombinant plasmid, the LB agar plates are supplemented with ampicillin and 5% of this sugar.
Sucrose
Immobilized Metal Affinity Chromatography (IMAC) works because the spin column resin is loaded with this specific metal ion.
Nickel
In the 8-well strip, the final well (Well 8) receives buffer and starch but no enzyme, serving as this essential experimental baseline.
Negative control
This lysis buffer contains a dye that turns the E. coli cell solution a shade of blue to indicate successful lysis.
Buffer P2
The gel electrophoresis tank is filled with this specific buffer—identified by a pink label in the kit—to allow the electrical current to flow.
TBE buffer
This specific gene encodes Levan sucrase and acts as a toxic marker in E. coli; it is eliminated from the plasmid during successful cloning.
SacB gene
Because it shares a structural similarity with histidine, a high concentration of this chemical is used in the elution buffer (NPI-500) to outcompete and release the purified protein.
Imidazole
During the serial dilution, transferring 10 µL of the enzyme mixture into 30 µL of sterile water creates this specific dilution ratio in each successive well.
1:4