DNA

What is the time frame for my synthesis?

We aim for next-day delivery on standard primers, and many modified primers under 100 bases. For primers over 100 nt, extended synthesis and deprotection times generally add 1-2 days. Availability on our two specialty machines is also a factor for turnaround time on modified or long primers.

Check out the DNA Service section for information regarding oligos with special modifications.

Reverse-phase HPLC purification adds anywhere from an extra day to a week, depending on the size of the order and HPLC system availability.

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Should I order crude or purified primers?
Perhaps our most common question. Various factors should be considered when choosing whether to order crude or purified oligos. These factors include the scale of the synthesis, the oligo length, the intended application for the oligo. In making your decision, it may be helpful to see how the efficiency of each round of base addition impacts the quality of oligo synthesis. The plot below shows the amount of full-length product expected from the synthesis of oligos of various lengths.

Below 40 bases

A majority of the material will be full-length, and using crude oligos is acceptable for most applications. Roughly half of our users exclusively order crude primers within this length range. Cartridge-purification of oligos below 40 bases removes most of the incomplete synthesis products resulting in purities from 85-95% depending on the given length. HPLC-purification improves the purity to above 95%.

Between 40 and 70 bases

More incomplete synthesis products, but most of the product will be full-length. Again, about half our users prefer to use crude oligos in this range. Cartridge purification removes most of the shortmers, giving 80-90% purity. HPLC improves the purity to above 90%.

Between 70 and 100 bases

About half of the material will be incomplete products. Many users still use crude oligos up to 100 bases, though it’s probably a good idea to at least cartridge-purify oligos of this length. Cartridge-purification removes roughly half the shortmers resulting in 70-85% purity. HPLC will afford purities around 90%.

Above 100 bases

Most of products are incomplete. We recommend purifying these longer oligos, but some users do still prefer them crude. Cartridge-purifiation will remove some shortmer products but they can still comprise as much as 50% of the material, especially for oligos above 150 bases. HPLC will afford better purity, but the amount of full-length material will generally not be above ~80%.
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How do I set up an account, or my login is no longer valid?
Please contact Esther Kim in the cores office at (801) 581-2425 or ekim@cores.utah.edu for all questions dealing with your account.
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Could my primer contain deletions?
Though rarely, some of our users have had instances where deletion products predominate in cloning experiments. In the cases where we’ve analyzed the oligos involved, mass spectrometry suggests that the oligos themselves are not the cause. Generally, the problem is resolved by altering cloning conditions (ie, cells, vectors) or re-designing the primers.
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How much buffer/H20 should I resuspend my primer in?
To determine the volume needed to dissolve dried primers, you will need to know the total amount of moles contained in the tube. The number of nmoles is supplied for purified primers, and primers are dissolved according to M=moles/L. For example, if you have 5 nmoles, add 50 ul of buffer for a final concentration of 100uM.

For crude oligos, the total Optical Density Units (ODU) are given. The total ODU can be converted to the number of nmoles using our online calculator. Simply log in to the ordering system and look up the order in question. Click the “show details” button, and input the total ODU value into the Absorbance field. Use a value of 1 for both the dilution and volume fields. The total nmole amount will be shown in the lower right.
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What yields can I expect?
It depends! Expect at least 4 nmoles for a standard cartridge-purified 40 nmol synthesis, 40 nmoles for the 200 nmol scale, and about 200 nmoles for the 1 umol scale.

However, since synthetic coupling efficiency drops off for longer primers, we cannot guarantee these yields for oligos above ~80 bases. In general a cartridge-purified 100mer gives 3-10 nmoles of purified product at the 40nmol scale, up to 20 nmoles at 200 nmol, and around 50-100 nmoles when synthesized at the 1 umol scale.

Yields on very long primers over 120 bases are even lower, and can also be sequence-dependent.
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RNA

What is the time frame for RNA synthesis?

Due to the presence of the large protecting group at the 2′ hydroxyl position, RNA require extended synthesis and deprotection times. Expect 2-5 days for completion of your RNA order, depending on the number of oligos ordered.
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What kinds of purifications are available?
Desalting is performed on all RNA oligos. We do not cartridge-purify RNA because of the conditions used to deprotect the 2′-hydroxyls. However, ion-exchange HPLC is available if better purity is needed.
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How should I store my RNA? Is it stable?
The best way to store RNA is to dry the oligo from a solution of water either by evaporation or precipitation. We provide the RNA in this dry form, and it should be stable for a year or more if kept at 4°C or lower. To store RNA in solution, the pH should be kept near neutral and nuclease-free. It’s best to keep it frozen in aliquots to minimize the number of freeze-thaw cycles. Under these conditions, RNA should be stable for at least 6 months in solution.
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If I send you my target gene sequence can you design my siRNAs for me?
Unfortunately, we do not design either RNA or DNA primers for users at this time. Please see our guidelines below if you are a first-time user ordering RNAi.
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Guidelines for ordering RNAi

  • All of our RNA synthesis services can be used to make siRNA. The “RNAi” service is distinct in that RNA synthesized in this manner must contain at least one 3′-deoxy overhang. The most common approach is to design sense and anti-sense 21mers, containing 19 RNA bases and 3′-dTdT overhangs. The 3′-deoxy overhangs allow for more efficient and less-costly synthesis (for 200nmole syntheses), and may enhance the efficacy of silencing.
  • To maximize stability, we provide the sense and antisense RNAi lyophilized and in separate tubes. The user anneals the two complementary strands to form a duplex molecule.
  • 200nmol “RNAi” syntheses generally give 40 -70 nmoles of desalted material.

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Peptide

What can I expect for yield and purity?

Peptides are usually delivered as the fractions collected after HPLC purification. Each fraction will have a different purity and amount. Yields vary with the sequence and length and are impossible to predict, but for simple peptides the user can expect the yield to be 10 to 20% of the listed scale. Almost all of the time, some of the fractions delivered will have better than 95% purity.
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What about peptide quantitation options?
Weighing a peptide after lyophilization may result in errors of up to 50% because of varying amounts of water left in the powder. The amount of water is difficult to control, even when the drying has been done thoroughly. Better ways to measure peptides are:

  • Amino acid analysis is the “gold standard” for measuring peptides. When you know the amino acid content, the test results in measurements of each amino acid present. AAA can be used for any peptide, regardless of amino acid content. The test is done in another lab, and we provide the free service of sending samples and calculating the results for peptides we have synthesized. We pass on to the customer the cost of the analysis charged to us.
  • The Ellman reaction is a very reliable measurement of reduced –SH groups on cysteine. If a peptide has a cysteine, we will provide amounts in the fraction table based on Ellman measurement. The reaction is easily done in the user’s lab as well. Ask us for a procedure.
  • The UV absorbance of peptides containing Tyrosine, and/or Tryptophan can be measured, and a theoretical extinction coefficient calculated. This is also easily done in the user’s lab.

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Is this a difficult peptide?

Peptides vary considerably in their difficulty to synthesize and purify. There are too many variables to describe here. Core facility chemists will be happy to advise users of foreseeable difficulties and suggest options. Please call to discuss or request quotations for complex syntheses.
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How long will it take?
We try to deliver peptides within two weeks after they are ordered. Since peptide synthesis is unpredictable we may need to repeat a synthesis to get the purity you need. If there is a backlog at the time of ordering, we will inform you of likely delays.
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