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RD Biotech / Molecular biology / Biologie MoléculaireRD Biotech / Services analytiques / Analytical servicesRD Biotech / Anticorps monoclonaux (mAb) / Monoclonal antibodiesRD Biotech / ELISARD Biotech / Culture Cellulaire / Cell cultureRD Biotech / Production de cellules / Cell production

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RD-BIOTECH

3 rue Henri Baigue

F-25000 Besançon

+33(0)3 81 53 88 37

rd-biotech@rd-biotech.com

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Molecular biology

Our molecular biology services are ideal if you wish to:
- To obtain quickly plasmid DNA in any quantity ?
- To make a customised vector ?
- To obtain a transcript or a protein where you don’t have the gene ?
- To purify a protein expressed fromyour own or a customised vector ?

RD-Biotech has available a complete panel of molecular biology services.
- By contacting and using RD Biotech:
- You will save time and money whilst you concentrate on the important aspects of your research
- You can take advantage of our scientific and technical expertise in molecular biology

PLASMID PRODUCTION

During recent years, plasmid purification has become a major activity of RD-Biotech.
We have amplified or created over 600 plasmids per annum giving us unrivaled expertise in producing plasmids for applications in research or preclinical studies.
Whether you need 1 mg of DNA for an R & D project or 500 mg of high quality endotoxin free DNA for in vivo or a sensitive in vitro applications, we can fulfill your requirements.

RD-Biotech is able to provide
- A 10 mg quantity in 3 days or 100 mg in 6 days!
- An endotoxin level guaranteed below 0.1 EU / µg DNA
- Large capacity to produce guaranteed quantities from milligrams to several hundreds od milligrams.
- Uniform batches of 90% supercoiled DNA
- We also offer Pre-and Post-production controls
- Selection of the right clone to be amplified, after restriction pattern analysis,
- Banking of your plasmid or clone for subsequent use
- Other services on request available are:

* Bacterial transformation using a strain of choice (Application and DNA dependant)
* DNA adjusted to a specific concentration
* Filtered DNA
* Aliquoting
* Determination of bacterial endotoxins (LAL / Limulus Amebocyte Lysate assay)
* Sterility tests


Applications

- Transfection of mammalian cells
- in vivo experiments (injection into animals)
- Preclinical programs
- Toxicology Studies
- Applications in Research & Development

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PREPARATION OF RNA

Preparation and purification of RNA from different sources (blood, cell pellets, biopsies...) for research or preclinical studies.
The procedure for extraction, purification and control of total RNA is optimized for the different starting materials.

1. Preparation of RNA from cultured cells
2. Purification of RNA from tissues
3. RNA isolation from whole blood, PBMC, platelets etc.
4. RNA preparation from plants...

- We have an automated platform for the preparation of total RNA, but manual method of extraction and purification of RNA is available

- Extraction of RNA from whole blood according to the PAXgene® technology

- Large production capacity ranging from 50 to 200 samples

- Possibility of preservation of RNA preparations (banking) for future use

- Reverse Transcription

- Quality control includes:

*checking the integrity of RNA by “RIN” (RNA Integrity Number)
*determination of total RNA content by Nanodrop® absorbance

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PREPARATION OF GENOMIC DNA

Preparation and purification of genomic DNA from different biological samples (blood, cell pellets, biopsies...) for applications in research or preclinical studies.
The procedure for preparation and control DNA is optimized for the starting material.

1. DNA preparation from whole blood, serum, peripheral blood mononuclear cells ‘PBMC’
2. Isolation of DNA from cultured cells
3. DNA preparation from plants…

- Platform for automated preparation of genomic DNA

- Extraction of DNA from whole blood using the PAX gene® technology

- Large production capacity ranging from 50 to 200 samples.

- Possibility of preservation of the DNA preparations (banking) for future applications (PCR, cloning, etc)

- Quality controls:

*Determination of DNA concentration by Nanodrop®
*Evaluation of DNA integrity by agarose gel electrophoresis.

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VECTOR CONSTRUCTION

RD-Biotech can construct expression vectors for applications in either prokaryotic or eukaryotic systems.

Our team of specialists in molecular biology designs the best strategy in accordance with your specifications.

Cloning service :

- Step by step
- A personal service with an assigned expert dedicated to the project monitors the project at each stage ensuring a satisfactory progress and outcome.
- An individual service: Each project is customised to best meet the needs of our customers: optimization of sequences, cloning strategy, choice of vector...
Each successive step of the project is performed only after validation by the customer (ie GO / No Go), for more flexibility and security
- Total confidentiality is assured.

Sequence preparation:

- Design of the sequence of interest
- Use of optimised expression systems dependant on the application (eg. addition of tagged labels, promoter sequences, insertion of specific cleavage sequences, etc)
- Shuttle and control vectors available
- Control of cloning by DNA sequencing

Standard cloning:

- Restriction enzyme digestion and purification of the insert
- Ligation and transformation into E. coli bacterial cells
- Screening of colonies
- Controls: Restriction pattern digests of recombinant plasmids by agarose gel electrophoresis
- Purification of plasmid DNA (Midiprep)
- DNA sequencing of the insert

Adapted cloning procedures:

- Mutagenesis
- Subcloning PCR: polymerase chain reaction, purification of PCR products
- Validation by DNA sequencing of the entire insert

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EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEINS

Using its multidisciplinary team, RD-Biotech offers a global platform for expression vector preparation and subsequent purification of recombinant proteins.
We can provide a standard service or a customised strategy tailored to your specifications. Our experts are at your disposal to propose the best route optimising progress of your project, in terms of schedule and cost.
RD-Biotech can perform all or part of the project, which can be broken down into in the following steps:

1. Project advice which may include scientific and technical analysis of bibliography

2. Analysis of protein configuration.

3. Construction of expression vectors

4. Feasibility and Pilot studies: testing various expression and purification conditions (searching the best cell line or bacterial strain whilst in parallel, testing different culture media conditions and temperatures etc), monitoring of each step so the customer can decide whether to proceed after analysing results (Providing a report and a GO / NO GO customer option)

5. Production and purification of the desired quantity.

6. Purified protein lots sent out with a report on production, purification and quality control


OUR SERVICE

- Prokaryotic expression systems: E. coli bacteria:

> A pilot study based on culture volumes of few ml
> Examination of induced proteins on an SDS-PAGE gel
> Analysis by Western-blotting (anti-tag or specific antibodies)
> Affinity column purification
> Scale up production: batches from mg up to several hundred milligrams

- Eukaryotic expression system: mammalian cells (CHO, HEK):

> Extensive experience in many cell lines: adherent cells or suspension cultures
> A pilot study in flasks or culture plates using small culture volumes
> Evaluation of protein expression by transient transfection
> Control of the expressed proteins: Western-Blotting, ELISA, flow cytometry...
> In the case of expression of recombinant antibodies (human, chimeric…) Assessment of productivity using the kit FastELISA RD-Biotech: Measurement of human immunoglobulin G in the culture supernatant
> Protein Purification: different systems depending on the specific protein (purification by affinity chromatography...)
> Assessment by Western-Blotting (anti-tag or specific antibodies) and SDS-PAGE gel electrophoresis
> Ability to scale up batch production:

* from a few milliliters up to several liters
* from a few millions up to several billions of cells

> Construction of a cell bank

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SEQUENCING OF MONOCLONAL ANTIBODIES

NEW! RD-Biotech has developed a platform for sequencing of monoclonal antibodies.

Pplate-forme de séquençage d'anticorps monoclonaux

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IN VITRO TRANSCRIPTION

in vitro transcription platform to generate RNAs from your plasmids or PCR products.

> Transcribed either in a sense or antisense direction
> Transcribed labeled or unlabeled
> Transcripts capped and / or poly-adenylated

- Preparation of biological material:

- Plasmid DNA:

Amplification of the plasmid to the desired quantity
Quality control: Quantification by Nanodrop® and agarose gel
electrophoresis after enzymatic digestion

- PCR Products:

Cloning vector with a promoter of interest (T7, SP6...)
Analysis by sequencing
Amplification and purification of plasmid DNA with quality controls

- Linearization of the plasmid by restriction enzyme.
- Purification of linearised DNA.
- Quantification by spectrophotometry and control by agarose gel electrophoresis.
- in vivo transcription procedure of sense or antisense transcript.
- Pilot transcription of 1 microgram DNA followed by transcription of the entire DNA provided.
- Purification of the transcripts and controls.
- Quantification of RNA obtained and examination by denaturing agarose gel electrophoresis.
- Control of RNA integrity by measuring RIN on a Bioanalyzer (Agilent).

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PCR AND REAL-TIME PCR

RD-Biotech can respond to problems related to quantification of genes, gene expression studies, analysis of the expression of different variants of a gene and identification of specific sequences...



> Classical PCR and RT-PCR platform:

- Development of operating conditions or application of a procedure validated by the customer
- Amplification
- Sequence determination of PCR products.
- Analysis of results and providing of a full report
- option: sample preparation prior to analysis



> Real-Time PCR platform:

The polymerase chain reaction in real time is a technique with many applications, based on continuous measurement of product produced by the PCRs enzymatic reaction. Each amplification cycle, the total amount of DNA or amplicon is measured with a fluorescent label. Obtaining the complete kinetics of the polymerization reaction makes it possible to obtain absolute quantification of the initial amount of target DNA present in the sample.

- Development of operating conditions or application of a standard procedure
- Absolute or relative quantification
- Procedures using Sybergreen® or TaqMan®


Development of qPCR Protocols: RD-Biotech offers an initial feasibility study, which if meets with the customer‘s approval, will be used for routine analysis.

1 - Feasibility Phase
- Bibliography study with analysis of published data
- Computer analysis of the sequence of interest
- Primer and protocol design
- Identification of house keeping genes
- Development of the qPCR procedure
- Amplifications on the number of samples
- Results, analysis and report writing.

2 - The routine analysis phase
NOTE: The quantity and quality of samples provided are the responsibility of the customer.

RD-Biotech can support the preparation of DNA and RNA samples from different biological materials.



> Routine use of the PCR or qPCR platform

- Conduct routine analyzes according to validated procedures. (Eg: Detection of mycoplasma by PCR)

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OTHER SERVICES

Our sales team, scientific and technical experts are at your disposal to offer you what is most appropriate for your application!

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