Generi Biotech - Compounds and Services for Molecular Biology

Frequently Asked Questions

You will find space for your questions, opinions and comments on this page. Should you have any questions, please do not hesitate to ask by filling in our electronic form. Our answer will be sent to the provided e-mail address and also (if you agree) published on our website.

If I would need to use blocking probe of 17-mer (particularly to avoid the amplification of WT sequence during PCR and vice versa) what is better and more stable 3` end modification for blocking the extension? Amino-group or dideoxynucleotide?

The 3` amino, 3` phosphate and 3` dideoxynucleotide may be used to block the extension according to our experience with 3` end extension blocked oligos. Unfortunately the 3` end dideoxynucleotide is the most expensive one and that is why the remaining two modification are using routinely. Based on our own experiments the 3` end amino modification is more stable than 3` end phosphate as the phosphate group may be hydrolyzed at certain conditions generating the free hydroxyl again ready for the extension.

Do you offer kits to detect the swine flu virus?

With regard to the threatening pandemic of the flu A/H1N1 (the so called swine flu), GENERI BIOTECH s.r.o. offers primer and probe sets for high-sensitivity detection using real-time PCR (qPCR). See here for more information >>

How does qPCR standard differ from PCR product cloning?

These two services differ in several aspects that are derived from their use. qPCR standards are primarily designated to be used in real-time PCR, and therefore it is assumed that the insert will be approximately 500 bp long, and the customer will obtain the plasmid in a concentration suitable to prepare the calibration curves. Plasmid selection depends on how the PCR product is prepared, and this selection cannot be influenced by the customer. As for cloning, PCR product length is limited only by technical capabilities of the cloning process (approximately 3 - 4 kb, depending on the plasmid type). The customer will obtain such a concentration of the plasmid that is suitable for various ways of use (further cloning, transfection etc.).

Do you offer random hexanucleotides?

Yes, random hexanucleotides (hexamers) are offered as a ready-made product. See here for more information >>

What are the excitation and emission wavelengths of fluorescent dyes for oligo marking that you supply?

GENERI BIOTECH synthesizes oligonucleotides marked by several commonly used fluorescent dyes (http://www.generi-biotech.com/oligo-catalogue-modified-5-end/). However, we can synthesize oligonucleotides marked also using many other, less common fluorescent dyes. Please find an overview of fluorescent dyes here including their spectral characteristics (excitation and emission maximums).

Please can you tell me how to get to the list of my purchase orders if I have no access password for My Oligo database?

Your code to enter the "My Oligo" database is included in every delivery note. To learn more visit: www.generi-biotech.com/my-oligo-info/

Please can you tell me whether and how to determine the real melting temperature (not calculated) of a dually marked probe and of primers?

In general, when Tm needs to be measured, this can be done by measuring the melting curve on the UV spectrophotometer with appropriate equipment (tempered cells with the programmable temperature program) or in the real-time cycler using SybrGreen. However, two complementary sequences are always needed. Nevertheless, our practical experience shows that although Tm calculated using the "nearest neighbour" method with cation concentration correction usually most matches the real value, real Tm (or Ta in PCR, respectively) must always be optimized in an experiment.

Sets to quantify housekeeping / reference genes of a renowned manufacturer reproduce genomic DNA; this means for my experimental setup that RNA must be treated with DNase. Do the reference genes you offer reproduce genomic DNA, as well?

All sets to quantify reference gene expression, offered by GENERI BIOTECH, are validated and it is assured that they do not reproduce genomic DNA. See here for more information >>

I have used a housekeeping gene in my experiments as recommended by a colleague from the neighbouring laboratory. However, in the present experiments we are not obtaining results that should be found.

In general, in experiments with gene expression quantification using real-time PCR for every experiment design, we recommend to determine the stable reference gene by means of choosing from multiple measured references genes (Article 1, Article 2).

At the same time, it is very advisable to use more than one reference gene for normalization also during the expression quantification procedure itself. (See here for more information >>).

I need oligos quickly and would like to order them using the website form. However, the order placement system at our workplace requires only official purchase orders placed through the administrative department with their own numbers. How should I resolve this?

Order the oligos using the web form, and enter the text "to be invoiced upon official purchase order receipt" - in the notes area - enter its number if you know it already. Oligos will be shipped with the delivery note only and the invoice will be issued upon receiving and matching your web order with the purchase order fro the administrative department.

Our workspace moved. Upon ordering from our new address for the first time, I obtained a new access code for MY OLIGO database. I would like to have access to all my purchase orders using a single code. Can anything be done about this?

Please send us information about your company (old and new delivery address) and MY OLIGO codes. Choose the code you want to cancel and the code you want to keep. We will unify the new and old code. The change will take place in a way to be reflected in MY OLIGO during several days. For information about situations where change of the MY OLIGO code is requested see here >>

What type of oligo purification should I choose?

GENERI BIOTECH offers standard (Sephadex), OPC and HPLC purification of oligonucleotides. In case of non-modified oligos up to 40 bases long and for common applications (PCR), standard purification is sufficient. For more sensitive applications, it is recommended to opt for OPC purification that is comparable to HPLC purification and shows the most favourable quality/price ration - for more information see here >>

Why do you not offer qPCR kits with SYBR Green I?

Based on our own experimental experience, GENERI BIOTECH offers only qPCR kits with hydrolyzation probes. This is due to the incomparably higher sensitivity and specifity of amplified product detection when oligonucleotide probes are used compared to SYBR Green I where detection of primer-dimer artefacts and other non-specific products of amplification takes place with high probability - See here for more information >>

Is it really necessary to purify the PCR product before sequencing?

Yes. PCR product contains non-incorporated dNTP and primers that must be removed before sequencing (dNTP from the amplification reaction changes the dNTP/ddNTP concentration ratio in the sequenation reaction). In cases the PCR product is isolated from the agarose gel, it also contains excessive salts present in the electrophoretic buffer.

Some commercial kits can be used, which operate on various principles: DNA bond to a little silicate membrane (for example, QIAquick PCR Purification Kit), separation of salts, dNTP and primers based on molecular weight using a gel (for example, CENTRI SPIN 20) or enzymatic single-step purification (for example, ExoSAP). The yield of the purification depends especially on the length of the PCR product, and the resulting concentration must be verified by means of electrophoresis - See here for more information >>.

What is the stability of your oligos and recommended storage?

Among members of specialized public, stability of oligonucleotides is frequently burdened with myths of their instability. GENERI BIOTECH verified using the experimental approach that oligonucleotides remained stable also if subjected to very poor treatment. A specific illustration of an experiment with results can be found here. It is recommended to divide oligos in aliquot parts upon dilution and to store them at -20°C - See here for more information >>.

Can you help me in some way to design the quantitative real-time PCR system?

Besides synthesis of primers and real-time PCR probes according to the sequence assigned by the client, GENERI BIOTECH also offers ready-to-use qPCR kits, complete design and optimization of custom-made qPCR kits and/or complex services of gene expression quantification are provided where all the steps are performed that are needed to obtain the results of relative or absolute quantification - See here for more information >>.

What is the purpose of internal positive controls in real-time PCR?

Internal positive control is another real-time PCR system (template, primers, probe) that is added to the reaction mixture together with the system to detect the target sequence (duplex reaction takes place). Monitoring of the positive control amplification is important to reveal false positive results that may occur due to PCR inhibition. GENERI BIOTECH offers specific internal positive controls or universal control - See here for more information >>.

May I have more modifications in a single oligonucleotide?

Yes, you may. GENERI BIOTECH provides modifications of oligos from our catalogue, and we can also prepare alternative variants of modifications based on the customer´s needs and/or their various combinations. With any "other than standard" purchase orders, it is recommended that you contact our personnel at and agree on the most practicable and economic solution of your requirement.

Do you also produce oligos with degenerated positions?

Yes, we do. GENERI BIOTECH provides degenerated oligonucleotides with mixed positions formed either by a simple mix of the bases or by inosine. The catalogue and pricelist of degenerated oligonucleotides can be found here.

Does the oligonucleotide sequence have an impact on the synthesis success?

Yes, it does. The synthesis yield is reduced together with increasing length of the oligonucleotide (the proportion of shorter incompletely synthesized chains rises), and it also depends directly on the sequence. Chains with a high representation of G and A that show lower coupling provide a markedly lower yield than chains with a high proportion of C and T. The lowest yield is shown by pure chains of polypurines - See here for more information >>.

What is the minimum and maximum length of oligonucleotide synthesis?

Oligos that are 2 to 99 bases long are synthesized as standard. Upon consultation, we can also prepare longer chains - please contact us at .

What does the term "Favoured Customer" mean?

The term "Favoured Customer" is used to describe our permanent customers who are entitled to receive a discount on all orders of oligonucleotides and probes. A general customer becomes a "Favoured Customer" upon ordering a quantity of oligos or probes at GENERI BIOTECH that corresponds to the so called cumulative consumption of 2000 bases. The status "Favoured Customer" then remains valid until the end of the year for the entire subsequent year - See here for more information >>

What type of oligonucleotide quality control do you apply?

MALDI-TOF quality control is used to control the quality of our oligos as standard. HPLC quality control is also provided for an extra fee - See here for more information >>

What if my oligo does not work? Will I get my money back?

Complaints of our products can be submitted within 6 months upon their receipt unless another period is stated for a specific product. If no apparent error is found on part of the customer, the complaint is resolved upon agreement with the customer by delivering a new product free of charge. In 2008, we received no complaints of any oligo or probe.

It is evident that the oligo quantity that I ordered does not correspond to the assigned scale. Why?

The synthesis scale as ordered provides only rough information of the resulting quantity. Absolute quantity of the product depends on the length of the oligonucleotide (longer oligonucleotide means more losses) and the assigned purification type (high losses are connected with HPLC purification) - See here for more information >>

How is the synthesis of oligonucleotides performed?

In vitro oligonucleotide synthesis is performed at GENERI BIOTECH using the so called phosphoramidite method whose principle consists in cyclic blocking of reactive groups of the growing oligonucleotide, which protect the molecules, and their subsequent revealing in order to connect another base - For more information see here >>