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HiPer® Bacterial Genomic DNA Extraction Teaching Kit (Column Based)

HiPer® Bacterial Genomic DNA Extraction Teaching Kit (Column Based)

HiPer® Bacterial Genomic DNA Extraction Teaching kit simplifies isolation of DNA from bacteria by the spincolumn procedure. Bacterial cells are grown in the medium till they reach log phase and are harvested by centrifugation. After harvesting, the bacterial cell wall is degraded by Proteinase K digestion and lysis. Following lysis, the DNA is allowed to bind to the silica-gel membrane of the HiElute Miniprep Spin column.

HiElute Miniprep Spin Column eliminates the need for alcohol precipitation, expensive resins, and harmful organic compounds such as phenol and chloroform, otherwise employed in traditional DNA isolation techniques. DNA binds specifically to the advanced silica-gel membrane while contaminants pass through. The adsorbed DNA is washed to remove trace salts and protein contaminants resulting in the elution of high quality DNA in the Elution Buffer provided with the kit.

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The data in Lane 1 and 2 demonstrates that highly purified bacterial genomic DNA has been obtained with no visible RNA contamination when electrophoresed on agarose gel. If RNA contamination is present, one would see a faint and smeary RNA band below the genomic DNA as shown in lane 3 since RNA being of lower molecular weight runs faster than the genomic DNA. RNA contamination is observed when the RNase treatment has not been carried out properly.

An absorbance of 1.0 at 260 nm corresponds to approximately 50 µg/ml of DNA. If the A260/A280 ratio is 1.6- 1.9, then the isolated DNA sample is considered to be pure. If higher A260/A280 ratio is observed it indicates the possibility of RNA contamination.



Cat No. Product Duration of Experiment Number of experiments that can be performed
HTBM008 HiPer® Bacterial Genomic DNA Extraction Teaching Kit (Column Based) Protocol: 1.5 hours; Agarose Gel Electrophoresis: 1 hour 10


HiPer® Multiplex PCR Teaching Kit

HiPer® Multiplex PCR Teaching Kit

Multiplex PCR is a widespread molecular biology technique for simultaneous amplification of two or more products in a single PCR experiment. This specialized PCR reaction employs different primer pairs in the same reaction for amplification of multiple target sequences in a single reaction mixture. This type of PCR often requires extensive optimization of annealing conditions compared to standard PCR systems using only two primers as an additional challenge of multiplex PCR is the varying hybridization kinetics of different primer pairs. Primers that bind with high efficiency could utilize more of the PCR reaction components, thereby reducing the yield of other PCR products. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.

There is an increasing demand for multiplex PCR techniques in Pathogen Identification, High Throughput SNP Genotyping, Mutation Analysis, Gene Deletion Analysis, Template Quantification, Linkage Analysis, RNA Detection and Forensic Studies.

 

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After performing agarose gel electrophoresis, one can check the simultaneous amplification of two PCR products. The amplicon size for GAPDH primers is 416 bp and that for 18s primers is 182 bp. The optimized conditions result in the multiple amplification of two PCR products of desired size.



Cat No. Product Duration of Experiment Number of experiments that can be performed
HTBM023 HiPer® Multiplex PCR Teaching Kit Protocol: 2 hours; Agarose Gel Electrophoresis: 45 minutes 5


HiPer® Transformation Teaching Kit

HiPer® Transformation Teaching Kit

For the incorporation of plasmid into a cell, bacteria must first be made “competent”. This process includes the treatment of cells with bivalent calcium ions in ice-cold condition. As a result small pores are formed on the cell membrane, which makes it permeable. The plasmid DNA may adhere to the surface of the cell and uptake is mediated by a pulsed heat shock at 42oC. A rapid chilling step on ice ensures the closure of the pores. These cells are allowed to propagate and selection of transformants can be done by growing the cells on a selective media which will allow only the plasmid containing cells to grow.

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On transformation of cells with pUC19 plasmid, antibiotic resistance is conferred on the host as this plasmid carries gene for ampicillin resistance. As a result, those cells that grow in presence of ampicillin are transformed cells. The transformed colonies are blue on X-Gal, IPTG plates due to α-complementation.



Cat No. Product Duration of Experiment Number of experiments that can be performed
HTBM017 HiPer® Transformation Teaching Kit Protocol: 4 days; Day 1: Preparation of media and revival of E. coli Host; Day 2: Inoculation of host strain from a single colony; Day 3: Protocol; Day 4: Observation and calculation of transformation efficiency 10


HiPer® Bacterial Gene Expression Teaching Kit

HiPer® Bacterial Gene Expression Teaching Kit

Plasmids that are used to carry foreign genes into a specific host are referred to as cloning vectors. It is important that a gene be inserted into the plasmid in the proper reading frame to ensure that the correct protein will be expressed. If the foreign genes are to be expressed in E. coli, one must use a bacterial expression vector. A bacterial expression vector contains all the elements required for transcription and translation which includes a strong bacterial promoter which can be recognized by the RNA polymerase and a ribosome-binding site called Shine-Dalgarno sequence. These expression vectors contain inducible promoters which can be controlled by the addition of an inducer. The most commonly used inducer for a bacterial expression system is Isopropyl-β-D-thio-galactoside (IPTG). IPTG induces the transcription of the gene coding for beta-galactosidase, an enzyme that promotes lactose utilization, by binding and inhibiting the LacI repressor. During a cloning experiment the lacZ gene is replaced with the gene of interest and IPTG is then used to induce gene expression. One advantage of IPTG for in vivo studies is that since it cannot be metabolized by E. coli its concentration remains constant. Plasmids carrying IPTG- inducible promoters are capable of expressing proteins at levels that exceed 30% of total mass of bacterial protein. Glutathione-Stransferase (GST) is often used as tags for proteins for expression and purification applications. An E. coli strain containing GST gene cloned into an expression vector having tac (lac + trp) promoter and lac I operator elements is grown to mid-log phase and induced with IPTG to allow expression of GST protein. The protein expression can be verified by SDS-PAGE by comparing the samples before and after induction.

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The GST protein band (29 kDa) can be seen in the after IPTG induction sample but barely visible in the before induction sample. This indicates an increased level of GST protein production.



Cat No. Product Duration of Experiment Number of experiments that can be performed
HTBM018 HiPer® Bacterial Gene Expression Teaching Kit Protocol: 3 days; Day 1: Preparation of media and revival of host strain; Day 2: Inoculation of host; Day 3: Induction and SDS-PAGE; Day 4: Observation and Interpretation 5