Basic methods

Quantitative real-time PCR experiments

GENERI BIOTECH commercially offers Real-time PCR SERVICE that is a type of service intended for those laboratories that use real-time PCR methods but not as their core techniques. Typically, they do not often find laboratory kits on the market that would meet their needs. Many laboratories using real-time PCR methods are not specialized with these techniques and usually they are not able to design their own real-time probes and/or find suitable real-time PCR conditions for them. Our service is inteded just for them.

Quantification of gene expression by QRT-PCR is a routine method utilized in many GB´s research activities. GB is able to cover all steps of quantification of gene expression experiments. In addition to real-time PCR SERVICE, GENERI BIOTECH offers synthesis of probes (P) and probe and primers design/synthesis (PPDS) - see real-time probes web site. Furthermore, GB can assist researchers in designing experiments as well as in all subsequent steps like RNA isolation, cDNA preparation, reference gene selection, measurment of samples or interpretation of results. We are also able to fullfill specific needs such as allelic discrimination (e.g. using LNA modified probes) or limited multiplex QRT-PCR for convenient and fast measurements (e.g. optimised duplex reactions of gene of interest with selected reference gene).

Typical aplication:

quantification of gene expression specific DNA detection (typically, microbial or viral detection) mutation and polymorphism detection (SNPs and e.g. FV Leiden, prothrombin and MTHFR) examples performed by GB

Related products: housekeeping genes, HC-QUEST, RISE

 

PCR related methods

PCR service offered on GB´s website is a service intended for laboratories that are currently using laboratory methods other than PCR and are not focused on molecular biology. Many laboratories that use PCR methods are not specialized with these techniques and typically they are not able to find kits that are useful for them. Many laboratories need to introduce into their routine practice a new molecular genetic technique but they do not have any ambition or even the facilities to develop it.

Simple cloning (sub-cloning)

GB offers cloning of selected DNA sequences into a plasmid, usually after PCR amplification. Cloned sequence is always verified by sequencing. A customer can provide us directly with his/her PCR product to be cloned or can only tell us sequence from GeneBank (or another bank) that he/she wants to be cloned. We can amplify the DNA sequence either with a Taq brand of polymerases or with proofreading polymerases. As a part of the cloning work we can carry out TA cloning, bacterial transformations and/or plasmid preps on demand. Using our own chemically competent cells, our transformation efficiency routinely achieves >10⁸.

Related products: Competent cells

 

Basic cell culture maintenance

In GB, an extensive amount of laboratory work is based on various cell culture techniques. GB offers its cell culture techniques, cell culture facilities and cell culture R&D solutions to scientists planning their cell line experiments however missing their own cell culture facilities.

Advanced methods available in GB:

Universal cell-based system for testing the nuclear uptake
Universal cell-based system for triplex forming oligonucleotides function testing
Original testing system to study efect of inhibitors of cyclooxygenases COX2 (group of drugs)

Nitrogen cell line storage

GB maintains its own cell line collection in liquid nitrogen. We offer both, long and short term storage of “guest” samples in the nitrogen bank. 

Phototoxic (VIS) assays

The phototoxic assays are carried out in human cell cultures as well as in the protozoan Paramecium culture. The equipment is tailored to use within visible light wavelengths (400 nm to 1100 nm). The cell viability is routinely measured by XTT assay, trypan blue exclusion or by method upon request.

Design and synthesis of specially modified oligonucleotides

Modifications are designed to be synthetically simple to fulfil specific conditions. They should enhance stability of TFO itself, they should not prevent from forming triplexes and should be able to conjugate with photosensitizers. We make an effort to perform synthesis of novel modifications of oligonucleotides that are supposed to form triple helix with target DNA duplex. Determination of stability of the formed triplex is necessary succesive step. Positively charged amidates are an example of such a modification.

Analysis and purification of oligonucleotides

HPLC and CGE analysis are applied in oligonucleotide quality control and purification (HPLC) to obtain highly purified oligonucleotides. The main aim is to find proper stationary phases, mobile phases and conditions, in which oligonucleotides differing in one or a few bases (or modified oligonucleotides vs. unmodified) are separated perfectly. An experience acquired by analysis of synthetic oligonucleotides can be used for analysis of metabolism of modified oligonucleotides. This type of analysis serves to elucidate metabolic pathway of modified oligonucleotides in cells.

 

Electrophoretic methods (agarose gel electrophoresis, PAGE, DGGE, SSCP, SDS-PAGE)

Sequencing analysis (sequencing plasmids, PCR products, sequencing projects incl. primer desing)

Fragmentation analysis (eg. cell lines characterization)