Oligonucleotides and primers are used in a variety of molecular biology methods, e.g. hybridization to detect specific DNA and RNA sequences or for amplification in PCR reactions. The accuracy and the purity of the oligonucleotide/primer sequence are critical for most applications. Primer or oligonucleotide synthesis in a lab requires not only an experienced person, but also expensive chemicals and a synthesizer. Therefore, several companies offer custom oligonucleotide/primer synthesis. Invitrogen, one of the largest companies, provides a variety of different options for oligonucleotide or primer synthesis at a reasonable price and with fast service.
Invitrogen’s website offers technical tools for choosing your oligonucleotide/primer sequences: Oligo Perfect™ Designer is the simple software for choosing primers, Vector NTI sequence analysis software offers more configuration options for primer design, while the web-based primer design Vector Designer™ is optimal for the construction of recombinant molecules. Designed oligonucleotide/primer sequences can be directly uploaded from the programs into the order form on Invitrogen’s website, thereby avoiding the errors possible when cutting and pasting sequences. Ordering is usually performed by uploading a single sequence into the basic form, adding the additional information required and sending the form via web, FAX or e-mail. Multiple sequences can be uploaded into either a list or a spreadsheet (high throughput, synthesis under the same conditions). Primers or oligonucleotides can be ordered in tubes (scale 25 nmole to 10 µmole, lyophilized) or for high-throughput, in 96- and 384- well plates (25 nmole to 1 µmole in water, TE, Tris-buffer; from 200 - 1200 µl volume).
Different purification options are available: desalted, purified over cartridge, HPLC or PAGE; the website provides information about the synthesis quantity and optimal length for each purifications. For most oligonucleotides/primers (between 20 and 30 bases) used in PCR amplifications, purification by desalting is sufficient. It is recommended to further purify longer oligonucleotides or those used for cloning through cartridge, HPLC or gel. Modifications can be added to oligonucleotides/primers in tubes either at the 5’ end (horseradish peroxidase, dyes, TET, amine, aldehyde, BODIPY with different wavelength), at internal sites (alternative or mixed bases) or the 3’ end (biotin, phosphate). A list of all modifications, their codes, excitation and emission wavelengths and extinction coefficients is provided on the website. Invitrogen also offers the possibility of ordering Stealth™ RNAi primers or siRNA duplexes for effective RNA silencing.
Delivery of the oligonucleotides/primers ordered before 5 pm (plate orders before 2 pm) take 2-3 days, while orders of purified or modified products can take approximately a week. The datasheet for each oligonucleotide/primer lists its purity and quantity. Protocols on the website describe the concentration calculations with examples of how to resuspend the lyophilized product; information on oligonucleotide/primer purification and spectrometry is also included.
We have used oligonucleotides/primers from Invitrogen routinely for PCR to screen mouse colonies. In addition, I have ordered several longer oligonucleotides (50 bases) in order to add linkers to genomic DNA in a PCR reaction. Since obtaining the accurate sequence was critical, I ordered them gel-purified, to eliminate shorter synthesis products or unincorporated bases. I never experienced any mismatches in my oligonucleotide/primer sequences after cloning and sequencing the products, indicating the high quality and accuracy of the ordered oligonucleotide/primer sequences. The oligonucleotides and primers are cost efficient, making it possible to order several primer pairs when establishing a new protocol for PCR screening.
For high quality and fast oligonucleotide/primer synthesis, I recommend using Invitrogen’s service. Ordering is easy, fast and the website contains information and FAQs about oligonucleotide/primer synthesis, yield guarantee, modifications, purification, design and quality, besides the information about ordering, delivering and pricing. Also, a video describes the synthesis facility and process.