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Custom Oligonucleotide Purification

Oligo Purification

All Gene Link oligos shorter than 40 mer do not require further purification if the applicaton is for PCR or sequencing. 20 mer oligo synthesized at a coupling efficiency of 99.5% will contain ~90% full length 20 mer and a mixture of truncated sequences of ~10%. As the length of the oligo increases, even at a coupling efficiency of 99.5%, the yield of the full length oligo is reducing. A 60 mer crude product will contain ~75% full-length oligo and similarly a 100 mer will contain ~60%. Purification is strongly recommended for oligos longer than 50 mer.



Oligo Size, Scale of Synthesis & Purification Guidelines
Product 50 nmol scale 200 nmol scale 1 umol scale 2 umol scale 10 umol scale 15 umol scale
Maximum Size 49 mer 99 mer 180 mer 250 mer 250 mer 250 mer
Purification Grade PAGE or RPC up to 35mer PAGE PAGE PAGE PAGE PAGE
All Gene Link oligos shorter than 40 mer usually do not require any further purification if the application is for PCR or sequencing.
Purification (price per DNA oligo, all prices in USD)
Product 50 nmol scale 200 nmol scale 1 umol scale 2 umol scale 10 umol scale 15 umol scale
Catalog No. (PAGE) 26-6400-77 26-6400-77 26-6400-55 26-6400-88 26-6400-44 26-6400-99
Gel Purification (PAGE) 75.00 75.00 150.00 280.00 1500.00 1800.00
Catalog No. (RPC) 26-6400-11 26-6400-11 26-6400-22 26-6400-23 26-6400-33 26-6400-34
Reverse Phase Purification (RPC) 30.00 30.00 90.00 170.00 750.00 900.00



Rush Oligo Purification Service

Certain experiments cannot wait for an extra day. When such is the case, order "Rush Oligo Purification Service". The custom oligo will be on fast track to be gel purified as soon as the oligos are synthesized and processed. This typically will save 2-3 days.



Rush Purification Service (Additional charge per DNA oligo, all prices in USD)
Product 50 nmol scale 200 nmol scale 1 umol scale 2 umol scale 10 umol scale 15 umol scale
Catalog No. (PAGE) 26-6400-77R 26-6400-77R 26-6400-55R 26-6400-88R 26-6400-44R 26-6400-99R
Rush PAGE Service Charge 35.00 35.00 50.00 65.00 250.00 350.00
Catalog No. (RPC) 26-6400-11R 26-6400-11R 26-6400-22R 26-6400-23R 26-6400-33R 26-6400-34R
Rush Reverse Phase Purification (RPC) Service Charge 10.00 10.00 15.00 20.00 40.00 60.00


Click here for RNA Oligos scale of synthesis and yield.

HPLC/RPC

HPLC and RPC (Reverse Phase Cartridge) purification methods yield purity of 85% to 95% depending upon the sequence, GC content and length of the oligonucletide. Reverse phase based HPLC fails above 40 mer as longer oligos are inherently hydrophobic and bind non-specifically.

Polyacrylamide Gel Purification (PAGE)

Purification by this method is considered the Gold Standard for oligo purification and yields 99%+ purity. Gel purification can be used for any length of oligonucletide (as compared to HPLC and RPC cartridges which are limited to oligonucleotides below 40 mer). Gel purification is strongly advised for all applications involving cloning of the product, such as mutagenesis and gene construction applications.

Comparison of Unpurified, RPC and Gel Purified Oligos

Polyacrylamide gel electrophoresis of crude, reverse phase cartridge (RPC) and gel purified oligos. Approximately 15 ug of crude unpurified oligo were loaded to show the truncated failure sequences. Approximately 8 ug of purified oligo were loaded. Lanes 1 to 3: 68 mer; lanes 4 to 6: 56 mer. Lanes 1 & 4: crude unpurified; lanes 2 & 5: RPC purified; lanes 3 & 6: gel purified.
Results: The above gel picture shows the lack of purification efficiency of RPC as compared to gel purification. Notice the remaining truncated oligo sequences that the RPC method failed to purify.

Comparison of Unpurified and Gel Purified Oligos

Polyacrylamide gel electrophoresis of crude and gel purified oligos in adjacent lanes. Lanes 1 & 2: 63 mer; lanes 3 & 4: 96 mer; lanes 5 & 6: 175 mer; lanes 7 & 8: 43 mer.
Results: At Gene Link we recommend gel purification of all long oligos and oligos used in cloning applications. Gel purification is the gold standard method of purification as the denaturing polyacrylamide gel resolution approaches single base and the major band is clearly visible to be excised and purified.

Oligo Size and Purification Recommendations
Length Page HPLC/RPC
8-40 mer Yes Yes
41-250 mer Yes No
All Gene Link oligos shorter than 40 mer usually do not require any further purification if the application is for PCR or sequencing.
Application Based Purification Recommendations
Application Purification
PCR and Sequencing Not Required
Cloning and Gene Construction Yes
Mutagenesis Yes
Modified Oligos Yes
Probes Yes


Scale of Synthesis & Yield

Click here for RNA Oligos scale of synthesis and yield.

Oligo Scale of Synthesis and Typical Yield of Unmodified DNA Oligos*
  Crude Desalted RPC Purified** Gel Purified
20 mer oligo
Typical Yield
30 mer oligo
Typical Yield
50 mer oligo
Typical Yield

Scale

A260 Units

nmols

mg

A260 Units

nmols

mg

A260 Units

nmols

mg

50 nmol

8-10 30+ 0.2-0.3 4-5 12+ 0.1-0.16 NR* [1-2] NR* [2-4] NR* [0.03-0.06]

200 nmol

20-25 80+ 0.6-0.8 8-12 24+ 0.26-0.4 4-6 8+ 0.13-0.2

1 umol

100-120 400+ 3-4 40-50 90+ 1.3-1.6 20-25 40+ 0.6-0.8

2 umol

200-240 800+ 6-8 80-100 180+ 2.5-3.1 40-50 80+ 1.2-1.5

10 umol

~1000 ~4000 ~30 ~400 ~900 ~10 ~200 ~400 ~60

15 umol

~1500 ~6000 ~45 ~600 ~1350 ~15 ~300 ~600 ~90

Purity & Yield

Purity is greater than 80%


depending on oligo sequence and structure. Refer to coupling efficiency table for oligo length dependent purity and yield.

No further purification required for PCR and sequencing applications.

Gel purification recommended for oligos above 50 mer and all applications involving cloning and mutagenesis.

**RPC is reverse phase purification using a cartridge; a substitue for HPLC.

Purity 85% to 95%


depending on oligo sequence and structure

Yield and Purity will be lower for sequences with high GC content

Not recommended for oligos longer than 35 mer.

**RPC is reverse phase purification using a cartridge; a substitue for HPLC.

Purity 98% to ~100%


depending on oligo sequence and structure

Yield will gradually decrease as length of oligo increases. Palindromes, hairpins and high GC content oligos and oligos containing stretches of 3 or more G's induce strong secondary structure and base stacking thus decreasing purity and yield.

*NR Not Recommended

*a. Typical yield stated in the table is for unmodified random sequence oligos. Reduction in yield is observed with high GC content oligos and those forming strong secondary structures.

b. Reduced yield is expected with modified oligos. The reduction percentage varies with modification type and number of sites. Typical reduction is 10%-20% per modified site.

c. Yield of 33µg/A260 unit for DNA oligos is calculated for an ~equimolar base composition. Long stretches of a single base or homopolymers will have variable yields. Example for homopolymeric 20 mer: dA(20) = ~25ug/A260 Unit; dC(20) = ~40ug/A260 Unit; dG(20) = ~32ug/A260 Unit and dT(20) = ~37 ug/A260 Unit



Post Synthesis Modifications Conjugation/Conversion (NHS, Thiol, Maleimide etc) Scale of Synthesis & Yield

Most oligo modifications are directly coupled using optimized automated DNA synthesis protocols that are suited for specific modifications. The final yield varies and are usually around 80% of the yield of the unmodified oligos. Multiple modifications and sites in the same oligo may lead to further reduction in final yield.
Certain modification (e.g Alexa fluorescent dyes, digoxigenin, etc) are not available for direct autoamted coupling chemsitry and have to be conjugated using post synthesis methods involving amino group to NHS (N - hydroxysuccinimide) ester, thiols to maleimide and other post synthesis conversions.
These post synthesis conjugation reaction kinetics yields ~60-85% final product and polyacrylamide gel purification is recommended. The yields guideline are listed below for these post synthesis conjugations.

Oligo Scale of Synthesis and Typical Yield of Post Synthesis Modifications*
(NHS, maleimide, base conversions, etc.)
  RPC Purified** Gel Purified
30 mer oligo
Typical Yield
50 mer oligo
Typical Yield

Scale

A260 Units

nmols

mg

A260 Units

nmols

mg

50 nmol

~2 ~4 ~0.06 ~1 ~2 ~0.03

200 nmol

~4 ~8 ~0.12 ~2.5 ~5 ~0.07

1 umol

~12 ~24 ~0.36 ~8 ~16 ~0.24

2 umol

~20 ~40 ~0.6 ~15 ~30 ~0.45

10 umol

~100 ~200 ~3.0 ~75 ~150 ~2.25

15 umol

~150 ~300 ~4.5 ~112 ~225 ~3.36

Purity & Yield

Purity 80% to 90%


depending on oligo sequence and structure

Yield and Purity will be lower for sequences with high GC content

Not recommended for oligos longer than 35 mer.

**RPC is reverse phase purification using a cartridge; a substitue for HPLC.

Purity 90% to ~98%


depending on oligo sequence and structure

Yield will gradually decrease as length of oligo increases. Palindromes, hairpins and high GC content oligos and oligos containing stretches of 3 or more G's induce strong secondary structure and base stacking thus decreasing purity and yield.

*NR Not Recommended

*a. Reduction in yield is observed with high GC content oligos and those forming strong secondary structures.

b. Reduced yield is expected with modified oligos. The reduction percentage varies with modification type and number of sites. Typical reduction is 10%-20% per modified site.

c. Yield of 33µg/A260 unit for DNA oligos is calculated for an ~equimolar base composition. Long stretches of a single base or homopolymers will have variable yields. Example for homopolymeric 20 mer: dA(20) = ~25ug/A260 Unit; dC(20) = ~40ug/A260 Unit; dG(20) = ~32ug/A260 Unit and dT(20) = ~37 ug/A260 Unit

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