Genotyping
RT-PCR
Molecular cloning
Plasmid design
Primer design
DNA/RNA/protein isolation
DNA sequencing
Antigen production
DNA/RNA/protein isolation
Why do you need DNA / RNA / Protein extraction?
Biological molecules extraction is a critical step in your experiments. You need to analyse molecules from your samples with a clear, reproducible and precise protocol. Our DNA / RNA / Protein extraction service will provide to you the molecular samples you need, extracted and quantified with a reliable protocol done by experimented people.
What do you need to provide?
For DNA: tissue or cells (frozen), blood and your sampling protocol. What kind of DNA do you want ? (genomic, mitochondrial, plasmid) and for what use ?
For RNA: tissue or cells (frozen at -70/-80°C), and your sampling protocol. What kind of RNA do you want? (mRNA, ribosomal RNA, mi/siRNA) and for what use ?
For proteins: tissues or cells (frozen), blood and your sampling protocol. What kind of protein do you want? (cytoplasmic, membranes, mitochondrial, nucleus, specific one) and for what use ?
What are the limits of the technique?
For all the molecules, sampling is the crucial step. Samples must be frozen as soon as possible, to block the action of DNAse, RNAse or proteinase. It is particularly crucial for RNAs. If those steps are not well done, the extracted molecules will have a poor quality.
What results we will provide?
We will provide the required biological samples, quantified and purified, ready to use for your experiments.
Genotyping
Why do you need genotyping ?
Genotyping consist to determine the genetic variation at a given position an individual possesses. Genotyping is used for (list is not exhaustive):
• Association studies (case/control)
• Linkage analysis
• Paternity test
• Forensic sciences
• ID profiling / Haplotyping
• Prediction from DNA profiles
• Molecular epidemiology
Genotyping can be done with PCR reactions. It is so fast and cheap to genotype a collection of hundreds of samples, to make a genetic variation bank, useful for any kind of analyses and a good complement to your protocol.
How we will proceed ?
We use the HRM (High Resolution Melting) technique to genotype. HRM genotyping consist to realize a PCR reaction of DNA fragments to genotype using a HRM fluorescent dye. During the DNA synthesis, the dye with be incorporated inside the double strand DNA structure. Once incorporated, the dye will emit fluorescence. At the end of the PCR reaction, using a temperature gradient, the DNA fragments will be denaturized, and the dye liberated, which will decrease the fluorescence. The HRM machine will detect the fluorescent decrease and then calculate the exact melting temperature of each DNA sample. As the melting temperature of DNA depend of its nucleotide composition, DNA samples can be classified in different groups, depending on their melting temperature. Then groups are identified according to their profile, to find what genetic variant they are carrying.
What do you need to provide ?
• Quantified DNA samples suitable for a standard PCR reaction (service available in our laboratory). For each genotyping, we need 100 µg of DNA. Piko96well say that it use 0.5ug of DNA for each reaction.
• Exact location of the position you want to genotype, and the FASTA sequence of the SNP region (+/- 500 bp around the SNP).
What are the limitations of HRM genotyping ?
• Because there must be only one difference between the DNA amplified fragments, the position to analyze must be the only one DNA variation.
What results we will provide?
We will give you the genotypes identified for each sample in an Excel sheet.
RT-PCR
Why do you need RT-PCR / qRT-PCR?
RT-PCR transcribes RNA into DNA. As it is an exponential amplification, you can specifically detect very small quantities of RNA in your samples with a RT-PCR for qualitative studies. It is the method of choice for RNA detection (with applications in virus/cells detection). RT-PCR is used for :
• Convert a specific RNA into DNA, and amply it
• Detect a specific RNA from cell, tissue, environment
Adding a real time quantitative step, RT-PCR becomes a qRT-PCR, which allow you to monitor in real time the DNA synthesis in your samples. That way you are able to determine the quantity of RNA in each sample before the amplification, for quantitative studies. qRT-PCR are often used to validate the most important results of DNA array, as this technique is more specific and introduce less false positives. qRT-PCR is used for :
• Quantify RNA in samples for quantitative studies
• Validate other transcriptomics results
What do you need to provide?
You need to provide quantified RNA samples with a RIN superior or equal to 7 (service available in our laboratory). If RIN are between 6 to 7, it is acceptable but could be more difficult to use. One reaction uses 50 ng of RNA.
What are the limitations of RT-PCR / qRT-PCR?
The RNA samples must have a good quality to work properly. RNA are more sensitive to degradation than DNA, so until they are reverse-transcripted, their manipulation is difficult. Despite its specificity, RT-PCR can give false positive due to unspecific amplification.
What results we will provide?
For each samples in RT-PCR, we will provide the resulted DNA products, quantified and purified.
For each sample in the qRT-PCR, we will provide the normalized quantity of RNA in the sample. Normalization will be done using the geometrical mean of 3 housekeeping genes (Vandesompele methods), not involved in the pathways related with the studied RNA.
Molecular cloning
Why do you need molecular cloning?
Molecular cloning consists to assemble recombinant DNA and replicate it within a host organism. Molecular cloning is used for:
• Recombinant protein production
• DNA library building
What do you need to provide?
You need to provide the FASTA sequence of the DNA fragment you want to clone and its source. In the case you want to provide your own vector, you need to provide it quantified and purified, ready to use for PCR reactions, with its map and FASTA sequence.
What are the limitations of the technique?
To properly work on the DNA cloning, we need to use restriction enzymes and PCR reactions. The DNA composition will limit the use of such enzymes and PCR primers.
What results we will provide?
We will give to you the final cloned DNA (quantified and purified), as well as the purified fragment and vector separately. We will also provide sequencing data to demonstrate that the construction is conformed to the request.
Plasmid design
Why do you need plasmid design?
For molecular cloning, you need to use a DNA plasmid to capture your recombinant DNA and replicate it in a host. For that, the plasmid should have special properties, depending on your experimenp, like for example expression system. You need plasmid design when the plasmid you need does not exist and needed to be synthetised.
What do you need to provide?
You need to provide your experimental protocol and conditions that we precisely know what are your needs, and what the plasmid should be.
What are the limitations of the technique?
Plasmid can have undesirable effects in the hosts, like toxicity. Also bacteria/yeasts needs to be transformed with the plasmid, which is a difficult step.
What results we will provide?
We will give to you the purified and quantified plasmid you need as a DNA solution. A plasmid map will also be provided. We can also transform cells for your experiment.
Primer design
Why do you need primer design?
PCRs are probably the most useful experiments in molecular genetics today. A good PCR reaction requires notably well-designed primers. A well-designed primer will able to make a clear and specific PCR reaction, avoiding false-positive and false-negative. But also, if you need to do many PCR reactions in your project with different primers, well-designed primers could allow you to run those different reactions at the same time, allowing you to save precious time. You need primer design service whe you want to be sure that your primers will be desiged by experienced people with PCRs.
What do you need to provide?
You need to provide the DNA fragment sequence (FASTA format, +/- 500 pb) you want to amplify with PCR, and information about the specie/tissue you are working with.
What results we will provide?
We will give to you the sequences of the best primers in FASTA format. You will able to order them with those specificities from your suppliers.
