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Selecting the right synthesis scale to order

To determine the appropriate scale for oligo synthesis consider:

  1. What is minimum purity level required for the technique(s) to be used?
  2. How much material is needed to complete the full investigation?
  3. Consult the adjacent yield tables to match the quantity of oligo required (listed by purification method) to the appropriate synthesis scale.

Although DNA synthesis often results in more material than the guaranteed minimum, if completing your experiments will require more than the minimum material then we highly recommend ordering a larger synthesis scale. For extended studies, we recommend that you freeze (–20 oC) aliquots the oligo, thawing a fresh aliquot for each stage of your investigations.

The minimum yields presented on the adjacent tabs are given in units of optical density (OD). For more information, see Calculations or contact our Technical Support team at 800-688-2248 or GenomicsSupport@eurofins.com.

Recommended purification based on the application

These are general guidelines for common applications, but customers are free to choose for themselves the appropriate purification. Available purification options are determined by oligo length, modifications, and synthesis scale. HPSF is a trademark of Eurofins Genomics and stands for High-Purity, Salt-Free, a column based purification method.

Application Oligo Salt-Free HPSF® HPLC PAGE
Standard PCR* and qPCR Unmodified X X    
Multiplex PCR Unmodified   X X  
Sequencing Unmodified   X    
Microarray Unmodified X X    
Modified   X X  
Blotting Unmodified X X    
Modified   X X  
cDNA Library prep Unmodified X X    
Primer Extension Unmodified   X    
Modified   X X  
454 Fusion Primer Unmodified   X X  
Modified     X  
Antisense Modified   X X  
Cloning (Chemical Linkers) Unmodified   X X X
End Labeling and FISH Modified     X  
Genotyping / SNPs Unmodified   X X  
Modified     X  
Kinasing Modified     X  
Primers with Restriction Sites Unmodified   X X  
qPCR Modified     X  
Crystallography Unmodified       X
Gel Shift Assays Unmodified       X
Gene Synthesis Unmodified       X
Mutagenesis Unmodified       X

 

Yield Specifications & Purity Recommendations

The tables in the tabs provide details of the guaranteed minimum yield for the unmodified oligo based on their synthesis scale of production. The yields of the modified oligos are highly dependent on the properties of the modifications included and, therefore, specific yields are not guaranteed.

The yields of the purified oligos are always lower than that of the standard salt-free oligos. Typically, if an oligo’s yield is 100 nmoles under salt-free conditions, HPLC purification will reduce the yield by 20 to 30 nmoles. In comparison, PAGE yields will be even lower as recovery of the oligo from the gel matrix is not efficient. A salt-free yield of 100 nmoles could be reduced to as little as ~10 to 12 nmoles of PAGE-purified oligo.

Final purity level of an oligo of ≥70 bases, or of an oligo with more than 2 modifications, is not guaranteed when employing a single purification method. For these oligo types, Eurofins Genomics strongly recommends using one of our 2-Step Purity options.

Available Purifications

Salt-Free Purity Yields

The standard salt-free purification is the default purity option for our oligos. For oligos shorter than 40 bases, salt-free purity is appropriate in many applications including routine PCR, DNA sequencing, microarray fabrication, and blotting.

Guaranteed minimum OD yields for salt-free purity for standard synthesis and ExpressOligos for unmodified oligos

Oligo length 10 nmole 25 nmole 50 nmole 0.2 µmole 1.0 µmole 10 µmole
6–10 0.5 0.75 1 5 10 120
10–14 1 1.5 2 5 15 150
15–19 1.5 3 3 10 30 300
20–45 2 3 6 25 45 450
46–60 2 3 9 35 90 740
61–90 NA NA 9 45 90 980
91–110 NA NA NA 15 135 By quote
110–125 NA NA NA 15 135 By quote

 

HPSF Purity Yields

High purity salt free (HPSF) purification is an inexpensive option that delivers excellent oligo yields and improved purity. Recommended for more sensitive applications such as qPCR or DNA sequencing, HPSF is available for modified oligos containing:

  • IUB Wobbles (degenerate nucleotide sites)
  • 5' Amino C6 Linker [Amino C6]
  • 5' Phosphate [Phos]
  • 5' Biotin-TEG [BioTEG] *

Guaranteed minimum yields (OD) for HPSF purity for unmodified oligos 

Oligo length 10 nmole 25 nmole 50 nmole 0.2 µmole 1.0 µmole
6–9 NA NA NA NA NA
10–14 0.5 0.5 1.5 2 5
15–19 0.5 1.5 3 5 10
20–60 1 2 4 10 35
61–80 NA NA 3 10 25
81–90 NA NA 3 5 20

HPLC Purity Yields

Guaranteed purity of 85% and above. HPLC is recommended for oligos used in more demanding techniques such as real-time qPCR, multiplex PCR, and cloning. Our purification scientists determine and employ the optimum HPLC technique for your specific oligo and select only the fractions containing the highest purity for delivery to your lab. Oligo purity of 85% for lengths above 70 bases, or of an oligo with more than 2 modifications, is not guaranteed when employing a single HPLC purification. For these oligos, please consider 2-Step purification to ensure high level purity.

The HPLC techniques employed include (1) reversed-phase HPLC purification that separates oligos on the basis of hydrophobicity or, (2) ion exchange HPLC which separates oligos on the basis of charge. 

Guaranteed minimum OD yields for HPLC purity for unmodified oligos

Oligo length 50 nmole 0.2 µmole 1.0 µmole 10 µmole
6–9 0.5 0.5 2 10
10–14 0.5 1 2 15
15–19 1 2 10 75
20–60 1 2 15 100
61–90 1 2 10 75
91–100   2 10 75

Guaranteed minimum OD yield for HPLC purified real-time qPCR Probes*

Oligo length 50 nmole 0.2 µmole 1 µmole
15–35 10 nmoles (~2.5 OD) 25 nmoles (~6 OD) 50 nmol (~12.5 OD)
36–45 5 nmoles (~1.25 OD) 12.5 nmoles (~3 OD) 25 nmol (~6 OD)

* Yield guarantees do not apply for real-time qPCR probes using modifications requiring NHS-ester coupling. Whenever in doubt, please inquire for information on specific DLP pairings.

PAGE Purity Yields 

Achieve purity levels of 90% and above with polyacrylamide gel electrophoresis (PAGE) purification. Because recovery of an oligo from the PAGE matrix is an inefficient process, the PAGE process results in low final yields. For that reason, PAGE is not recommended for routine purification.

Guaranteed minimum OD yield for PAGE purity of unmodified oligos

Oligo length 50 nmole 0.2 µmole 1.0 µmole
20–45 1 4 20
46–60 1 4 20
61–80 1 3 10
81–90 1 2 10
91–100 NA 2 10

Expected yield for a PAGE oligo that includes one modification would be ~60% of the value listed.

2-Step HPLC Yields

This 2-step purification process uses reversed-phase HPLC followed by anion exchange HPLC, allowing a two-dimensional purification based on both hydrophobicity and charge. This purification method is highly recommended for all oligos ≥70 bases.

Guaranteed minimum OD yield for 2-Step HPLC purity of unmodified oligos

Oligo length 0.2 µmole 1.0 µmole 10 µmole
6–9 0.5 1 10
10–14 0.5 1 10
15–19 1 5 15
20–45 1 5 15
46–60 1 5 15
61–69 1 5 15
70–80 1 5 15
81–90 0.5 2 10
91–100 0.5 2 10
101–125 0.5 2 By quote

* Yields in parentheses refer to a single 5’ or internal modification. 3’ modification yields may be lower.

2-Step PAGE Yields

This 2-step purification process uses reversed-phase (RP) chromatography followed by PAGE, allowing a two-dimensional purification based on hydrophobicity and molecular weight. This purification level is highly recommended for oligos ≥70 bases used in applications that require PAGE purification and for all oligos used in applications that require the highest purity levels, such as crystallography and gel-shift assays.

Guaranteed minimum OD yield for 2-Step PAGE purity

Oligo length 0.2 µmole 1.0 µmole
6-19 NA NA
20-45 1.0 2.0
46-60 1.0 2.0
61-69 1.0 2.0
70-80 0.5 1.0
81-90 0.5 0.5
91-100 0.5 0.5
101-125 0.5 0.5

Possible yield for a 2-Step PAGE oligo that includes a simple modification would be ~60% of the values listed.