MGH   
CCIB
Home > Synthesis > current
 
DNA Synthesis: Frequently Asked Questions

 

I would like to use your oligonucleotide synthesis services. How do I set up a new account?

To create a new account, please click the Create New Account link (to be found on the top of all the pages of our website) and fill out the required fields of the Registration Form. To complete the registration process, click on the Register My Account link. Your account will be active immediately after registration, and you are ready to submit your first order!

Which synthesis scale should I order?

If you are planning to test your oligonucleotide in small-scale PCR-based experiments, the 50 nmole synthesis scale is most likely sufficient. If you are ordering an oligonucleotide that is used routinely for medium- to high-throughput experiments or an oligonucleotide that is longer than 50 bases, you may want to request the 200 nmole scale.

What is the synthesis scale?

The synthesis scale represents the amount (nmole) of starting material for the first base at the beginning of the oligonucleotide synthesis (i.e. the amount (nmole) of the first base attached to the solid support). The scale does not indicate the expected yield. The overall oligonucleotide yield depends on the average coupling efficiency of each base and on the number of couplings (length of oligonucleotide). Additional losses in yield may occur during post-synthetic processing, transfer of material and quality control.

What is coupling efficiency?

Coupling efficiency is a way of measuring how efficiently the DNA synthesizer is adding new bases to the growing DNA chain. It is influenced by oligonucleotide sequence, oligonucleotide length and modification type. During DNA synthesis, the maximum coupling efficiency obtainable is normally around 99%. This means that at every coupling step approximately 1% of the available bases fail to react with the new base being added.

How does the CCIB DNA Core quantitate oligonucleotides?

The use of a spectrophotometer is an efficient method for determining the actual yield. We carefully quantify oligonucleotides by measuring their optical density (OD) at a wavelength of 260 nm.

How does the CCIB DNA Core check the quality of the synthesized oligonucleotides?

All of our research-grade oligonucleotides are quantitated via UV absorption to verify successful synthesis and to determine the exact yield. The purity of our products is not routinely tested but we do monitor quality by sending out randomly selected samples to a trusted third-party vendor that will perform QC checks via capillary electrophoresis (CE) and/or mass spectroscopy.

How can I calculate the concentration of my sample?

For each oligonucleotide, a specification summary (including sequence, OD260, molecular weight, and volume) is provided and can be downloaded via our website. This information can be used to calculate both the mass concentration and the molar concentration of the oligonucleotide in solution.

1. Mass Concentration (µg/ml)

The equation is:

c = A * e / V

c = mass concentration of nucleic acid (µg/ml)
A = absorbance at 260 nm
e = extinction coefficient (~33µg/ml for single stranded DNA, with an equal mixture of each of the four bases) V = volume of nucleic acid solution (ml)

Here is an example:

If an oligonucleotide solution has 28 OD's in 500 µL, the calculation would be:

28 * 33 / 0.5 = 1848 µg/mL

2. Molar Concentration

The equation is:

c = A * e / MW / V

c = molar concentration of oligonucleotide (uM, µmoles/l)
A = absorbance at 260 nm
e = extinction coefficient (~33µg/ml for single stranded DNA, with an equal mixture of each of the four bases) MW = molecular weight of oligonucleotide (g)
V = volume of oligonucleotide solution (l)


Here is an example:

If an oligonucleotide solution has 28 OD's in 500 µL, and the molecular weight of the oligonucleotide is 9000 grams, the calculation would be:

28 * 33 / 9000 / 0.0005 = 205.33 µmoles/l (205.33 µM) or

28 * 33 / 9000 / 0.5 = 0.205 µmoles/ml (205.33 µM)

How are the oligonucleotides supplied?

Our oligonucleotides are supplied deprotected and resuspended in nuclease-free sterile water (neutral pH!). Upon special request, oligonucleotides can be provided in lyophilized form. Oligonucleotides are usually packaged in 2-ml screw cap micro tubes (Sarstedt brand). If you place an order for 24 or more oligonucleotides, you can request packaging in 96-well format.

What are the recommended storage conditions for oligonucleotides?

Our oligonucleotides are usually resuspended in sterile nuclease-free water (neutral pH!), at a concentration of 1 µg/ml or greater, and are stable for 6-12 months if stored at -20°C. Repeated freeze-thaw cycles have to be avoided to prevent degradation. Oligonucleotides with dye modifications have to be protected from light at all times.

Lyophilized oligonucleotides (provided upon special request) are chemically stable for at least one year if stored at -20°C.

I ordered my oligonucleotide in lyophilized form. How do I reconstitute it?

Upon receipt, please spin down the tube containing the oligonucleotide as part of the product might stick to the lid. Stock solutions should be prepared by resuspension in nuclease-free sterile water (neutral pH!) or in 1X TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5-8.0) at a concentration of 1 µg/ml or greater and stored in small aliquots at -20°C. We do not recommend the use of deionized water since the pH of the water is often slightly acidic and can cause hydrolysis of the oligonucleotide.

How do I make a 100 µMolar stock solution of my lyophilized oligonucleotide?

The CCIB DNA Core provides quantitative data on your oligonucleotide using nmoles, optical density units (OD) and micrograms. This allows simple preparation of stock solutions for whatever units you prefer. A general rule states that for any oligonucleotide, the number of nmoles multiplied by 10 will give you the amount of solvent (in microliters) to add for a 100 µM stock solution. 100 µM is also equal to 100 pmol/microliter. In general, oligonucleotides will dissolve in this volume - when vortexed - within a few minutes.

What is the longest oligonucleotide that the CCIB DNA Core will synthesize?

Our facility utilizes a BioAutomation MerMade 384 Well DNA/RNA synthesizer that is capable to manufacture oligonucleotides with 120-130 bases. Since more than 95% of our orders request oligonucleotides shorter than 60 bases, we opted to synthesize oligonucleotides up to a maximum length of 75 bases. This enables us to fulfill the increasing demand for our DNA synthesis services, to achieve greater operational efficiency and to maintain our rapid turnaround for our standard products.

To meet all of our customers' research needs, we are now providing very long oligonucleotides (76-100 bases) - including PAGE purification option if desired - through outsourcing to IDT. Outsourced oligonucleotides are discounted from IDT's list price and receive free shipping to our core facility.

Does my oligonucleotide have a phosphate on the 5 or 3 end?

No, it doesn't. All of our custom oligonucleotides are synthesized with a hydroxyl group on both the 3' and 5' ends. However, if requested, we can synthesize your oligonucleotide with a 5' and/or 3' phosphate.

What oligonucleotide modifications does the CCIB DNA Core offer?

The CCIB DNA Core routinely incorporates the following oligonucleotide modifications at the time of synthesis: 5' Biotin, 5' 6-FAM, 5' HEX, and 5' Phosphate. In these cases, the cost for the modifying reagent will be added on to the standard synthesis fee. If you would like to order an oligonucleotide with another type of modification, please contact us for availability.

Can I order an oligonucleotide with degenerate (mixed) bases?

Yes. Our team offers the synthesis of DNA oligonucleotides with equimolar mixtures of two or more different bases at a given position within the sequence. Synthesis scales of 50 nmole and 200 nmole are available. No extra charges apply. When ordering an oligonucleotide with mixed or degenerate bases, please use the standard IUB (International Union of Biochemistry) codes to represent the degenerate base.
*Please note that our synthesis equipment limits the number of unique degenerate bases to three. Here is an example: ACGTANNNACGTSS ACGTRRRACTTC

Code

Base Mix

Code

Base Mix

Code

Base Mix

R

A,G

Y

C,T

M

A,C

K

G,T

S

G,C

W

A,T

H

A,T,C

B

G,C,T

V

G,A,C

D

G,A,T

N

G,A,C,T

 

 

What purification methods are available?

For all of our in-house oligonucleotide products, we offer desalting as an add-on service. Our cartridge purification method efficiently removes residual by-products from the synthesis, cleavage and deprotection procedures salts (i.e. salts, base protecting groups and short failure sequences up to 6 bases). Desalted oligonucleotides can be used for standard molecular biology applications including PCR, RT-PCR, DNA sequencing, primer extension, hybridization, and antisense technologies.

For long unmodified oligonucleotides (76-100 bases), we recommend PAGE purification. For this service option, both synthesis and purification will be outsourced to IDT.

Can you provide guidelines for designing proper sequencing primers?

Yes, we do provide general guidelines for the proper design of sequencing primers. Please refer to our Sequencing Guidelines for details.

Please be aware that no set of guidelines will always accurately predict the success of a primer. Some primers may fail for no apparent reason, and primers that appear to be poor candidates may work well for automated sequencing.

What kind of chemistry does the CCIB DNA Core use?

The CCIB DNA Core routinely uses solid phase synthesis and performs phosphoramidite chemistry.

Where can I learn more about the BioAutomation MerMade 384 Well DNA/RNA synthesizer?

Please refer to this link to learn more about the features of the BioAutomation MerMade 384 Well DNA/RNA Synthesizer.

What is the turnaround time for an oligonucleotide order?

Typical processing time for standard in-house oligonucleotides (with up to 75 bases; no modifications; -/+ desalting) is 24 hours. If the total customer order volume exceeds our current production capacity, however, samples will be entered into the queue and processed in the subsequent run (on the next day). Most orders will be available for pick-up at designated locations within 48 hours (excluding Friday orders). Upon special request, oligo orders can also be picked up within 24 hours directly at our core facility (38 Sidney Street, Suite 100, Cambridge, MA).
If you have chosen one of our outsourced synthesis/purification services, all orders are delivered to the CCIB DNA Core based on standard IDT shipping procedures. Upon their arrival at the core facility, all orders will be delivered to our remote pick-up locations via Partners Courier Service.

Please contact us for more information regarding the turnaround time of lyophilized, modified and/or long oligonucleotides (> 76 bases).

Where can I pick up my oligonucleotide?

We offer a convenient and free delivery service to designated locations (via Partners Courier Service). For a detailed list of our remote pick-up locations, please see here. Upon special request, completed oligo orders can also be picked up at our facility (38 Sidney Street, Suite 100, Cambridge, MA 02139).

How much does custom oligonucleotide synthesis cost?

Please review our pricing details here.