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Custom Oligonucleotide Synthesis

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.

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
Oligo Scale of Synthesis and Typical Yield of Unmodified DNA Oligos*
  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

4-5 12+ 0.1-0.16 NR* [1-2] NR* [2-4] NR* [0.03-0.06]

200 nmol

8-12 24+ 0.26-0.4 4-6 8+ 0.13-0.2

1 mmol

40-50 90+ 1.3-1.6 20-25 40+ 0.6-0.8

Purity & Yield

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

Purification

All Gene Link oligos shorter than 40 mer do not require and further purification if the applicaton is for PCR or sequencing. Gene Link recommends gel purification of oligos longer than 50 mer and all oligos destined to be cloned.

Scale of Synthesis Price ($)/purification

Product

Catalog No.

50 nmol

200 nmol

1 mmol

2 mmol

10 mmol

15 mmol

Gel Purification

26-6400-XX

75.00

75.00

150.00

280.00

1500.00

1800.00

Reverse Phase Cartridge

26-6400-XX

30.00

30.00

90.00

170.00

750.00

900.00

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