Frequently Asked Questions - Oligonucleotide Synthesis

Product & Services

Do you also offer the synthesis of MGB-probes?

Yes, we do offer MGB probes with different dyes.
The modification (3’ MGB-Q530) can be ordered directly through our webshop.

I work with degenerated oligos and need an unequal base distribution. Can you synthesize such oligos?

Yes, Microsynth has lot of experience in degenerate oligos for various applications. We can offer you degenerate oligos with following characteristics:

Product	Applications	How to order?
Degenerated Bases (IUB Hand Mixed Synthesis Reagents)	NGS, barcoding and mutagenesis applications where equal distribution of degenerate bases is critical.	requires a specific quote
Degenerated Bases (Custom Hand Mixed Synthesis Reagents)		requires a specific quote
Degenerated Bases (Standard IUB Wobbles)*	All other applications where equal distribution is less important or the sequence space is too big to be covered with one standard synthesis i.e. some SELEX applications	directly via webshop

*Bases are automatically mixed by synthesizer.

Do you design primers? If yes what is the price?

Yes, we perform primer design for customers (standard PCR primers, qPCR assays as well as siRNAs). The price is depending on the request. Up to 5 primer designs per day and customer are free of charge. If the design is special or significantly more primer designs are needed, we will give you an offer about the pricing. We prefer NCBI-Accession numbers for primer designs, but also accept sequences in plain text / fasta format.

Are your primers and probes compatible with commercially available supermixes for use in digital droplet PCR?

Yes.

Microsynth primers can be further used in EvaGreen or probe based assays (e.g. drop-off assays for mutation screening).

Order Related Questions

I want to order control siRNA. How should I proceed?

Order by email the control siRNAs indicating which and how many aliquots of control siRNA you need (oligo.support@microsynth.ch).

It is also possible to add presynthesized control siRNA to your order of custom siRNAs. During the ordering of your siRNAs you can specify the name(s) and number of aliquots of the required control siRNAs in the field "special comment".

I want to order oligos in 384 well plates. Is this possible?

Yes, we can deliver oligos in 384 well plates. Please download our excel order sheet under Upload entry in the webshop. Paste your sequences in the file and send the excel file by email to oligo.support@microsynth.ch. Do not submit the excel sheet directly in the webshop. We will come back to you with an offer and more details about your order.

How can I order hybrid oligos (DNA/RNA/2’-O-Methyl-RNA)?

Replace the different DNA/RNA or 2’-O-Methyl-RNA Bases in your sequence by the internal modifications 5, 6, 7 and 8 (e.g. TTAGCrAArGTTrUrC -> TTAGC5A6TT78). Login our webshop and enter your sequence into the sequence field. Define the modification in the drop down menu for 5, 6, 7, 8 (either RNA/DNA or 2’-O-Methyl-RNA bases).

I want to order oligonucleotides in an 96 well-plate, but I need a special layout. How can this be done?

You can shift the position of oligonucleotides in the 96 well plate by placing empty position. To accomplish that enter an oligo dataset with the following properties:

i. Oligoname: Leerposition
ii. Scale: Genomics
iii. Purification: Desalted
iv. Sequence: TT

Is there a minimal amount of oligonucleotides, that I have to order, to receive the oligo order in an 96 well plate?

The minimum amount of oligos to receive an 96 well plate is 40 oligonucleotides.

I want to order oligos longer than 150 DNA bases. How should I proceed?

We would like to discuss the proposed experiment/application with you beforehand to guarantee the best possible outcome. Such long oligos are not recommended for direct cloning experiments but can be applied for PCR amplification and subsequent cloning or as templates (positive control) for qPCR assays.

Oligos longer than 150 DNA bases can be ordered in the 0.2 µmol synthesis scale as well as in desalted quality only. When ordering such long oligos through our webshop, please proceed as follows:

Shorten your sequence to 100nt
Choose “others” for 3’ modification
Follow the further instructions and submit your order (due to the 3’ modification “others” the production will be halted until we got the entire sequence from you)
When receiving the order confirmation email from Microsynth, reply to this email by including the entire sequence information (others = entire DNA sequence). Do not forget to indicate the 5’ and 3’ end of the sequence in the email.

I want to order an oligo with an internal dye modification. How should I proceed?

Usually, internal fluorescence modifications are incorporated as dT-compounds (e.g. FAM-dT). The ordering procedure for this type of modifications is very simple:

Select a “T” DNA nucleotide within your sequence and replace the “T“ by “5”
Select your preferred dye (e.g. FAM-dT) in the drop-down menu under “Inner Modification (5=...)”
Follow the further instructions and submit your order

Should you need the internal dye modification on a different nucleotide please get in contact with us. You will need to add the modification using “others” and inform us on the actual modification.

How best to order oligos with different production times?

Partial delivery of oligos from one single order is not possible. If your order comprises several oligos with different delivery periods, you will receive all items as a single consignment as soon as all products are ready for shipment. Please use distinct orders, if delivery speed is critical.

Microsynth offers 3 different levels of purity for siRNA to be used in vitro. Can you consult me which type of purification I should use for which experimental setup?

Initial screening using different siRNAs: for such an application our customers usually use desalted siRNA which has undergone cartridge purification. Herewith purity levels of ~80 % are achieved which is fairly enough for initial screening purposes.

Advanced screening or for use in cell cultures: if your experiment becomes more sophisticated and hence more expensive, we usually recommend to select HPLC (~85%) or even PAGE purification (~95%).

Does Microsynth have siRNA for in vivo experiments?

Yes, Microsynth does offer custom siRNA for use in animal experiments. The processing options include various HPLC purification methods, counter ion (Na+) exchange and sterile filtration.

General Technical Questions

I would like to know the 5‘ and 3’ moiety of the standard DNA oligos you provide. Are they 5’OH?

By standard, there is a OH-group at the 5’ and 3’ of an oligo.

What is the stability of DNA/RNA oligos and how should they be stored?

More information on DNA oligo handling, storage and stability can be found here.

More information on RNA oligo handling, storage and stability can be found here.

How does Microsynth calculate the molar extinction coefficient of oligonucleotides?

Molar extinction coefficients of oligonucleotides are calculated according to the „base composition model“. [1-3] It includes the absorption of the individual nucleobases and potential base modifications and/or dyes. More accurate values for unmodified oligonucleotides are obtained from the „nearest-neighbor model“, which considers stacking effects of adjacent nucleobases. According to our calculations this model reduces the extinction coefficient approximately to 90 % compared to the base composition model. Therefore the extinction coefficients of oligonucleotides are estimated according to the below equation.

Tataurov A.V., You Y., and Owczarzy R. (2008) Predicting ultraviolet spectrum of single stranded and double stranded deoxyribonucleic acids, Biophys. Chem. 133, 66-70.
Fasman, G.D. (Ed.) (1975) Handbook of Biochemistry and Molecular Biology, Volume 1: Nucleic Acids, pp 589, 3rd edition, CRC Press.
Cavaluzzi M.J., and Borer P.N. (2004) Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNA, Nucleic Acids Res. 32, e13.