Scale of synthesis refers to the amount of starting CPG (controlled-pore glass) support-bound monomer used to initiate the DNA synthesis, not the amount of final material synthesized. As an example, a 20 mer synthesized at a 200 nanomole scale of synthesis will produce approximately 80 nanomoles. The losses occur during synthesis, post-synthetic processing, transfer of material, and quality control.
Do I need to have my oligo purified?
It depends on whether or not modifications are requested and what the application will be. Failure sequences may be generated both during the synthesis and post-synthesis processing. We recommend that all modifications be purified either by cartridge or HPLC. For recommended purity and scale (based upon application), please see Table I.
Application
Scale of Synthesis
Purification
Antisense studies
1 µM
RP1
End labelling
.2 µM
HPLC, PAGE
Gel shift assay
.2 µM
HPLC, PAGE
Gene synthesis
.2 µM
PAGE
Hybridization
.2 µM
Desalt, RP1
Kinasing
.2 µM
HPLC, PAGE
Mutagenesis
.5 µM, .2 µM
HPLC, PAGE
PCR
.5 nM, .2 µM
Desalted
Qualitative PCR
.5 µM, .2 µM
HPLC, PAGE
RT-PCR
.5 µM, .2 µM
Desalt
Sequencing
.5 µM, .2 µM
Desalt
Modified bases and chemical linkers
.5 µM, .2 µM
RP1
Reporter groups (biotin, DIG or fluorescent dyes)
.5 µM, .2 µM
RP1, HPLC
How much do I get or what scale of synthesis should I order?
Table II
Estimated Yields for Different Purifications and Turnaround
Scale of Synthesis
Purifications
Yield
Turnaround
0.02 uM 24 hour service
Desalt
2-3 OD
24 hours
0.02 uM 48 hour service
Desalt
2-3 OD
48 hours
0.05 uM 24 hour service
Desalt
3-10 OD
24 hours
0.05 uM 48 hour service
Desalt
3-10 OD
48 hours
0.05 uM 2-3 day service
Desalt
3-10 OD
2-3 days
RP1
2-5 OD
2 additional days
PAGE
0.5-1 OD
3 additional days
HPLC
1-2 OD
3 additional days
0.2 uM 24 hour service
Desalt
10-20 OD
24 hours
0.2 uM 48 hour service
Desalt
10-20 OD
48 hours
0.2 uM 2-3 day service
Desalt
10-20 OD
2-3 days
RP1
3-7 OD
2 additional days
PAGE
1-2 OD
3 additional days
HPLC
2-5 OD
3 additional days
1.0 uM 24 hour service
Desalt
20-50 OD
24 hours
1.0 uM 48 hour service
Desalt
20-50 OD
48 hours
1.0 uM 2-3 day service
Desalt
20-50 OD
2-3 days
RP1
5-10 OD
2 additional days
PAGE
3-5 OD
3 additional days
HPLC
5-10 OD
3 additional days
What do I re-suspend my oligo in and what concentration should I make it?
Purified water, PBS or any biological buffers are acceptable as diluents. The recommended diluent volume is 100 µl - 1 ml, the concentration depending on the application to be used and the yield of the resulting product. Standard concentration for PCR primers is 0.1 mM.
How do I determine my concentration?
Concentration is determined by measuring the OD260 of the diluted oligo. Prepare a dilution of the resuspended oligo and measure the OD260 . Determine the concentration as follows:
(µg or pmoles/ OD260 ) X dilution factor = final concentration / mL.
How stable is my oligo once I have resuspended it?
If sterile diluent is used to resuspend the oligo, it will be stable at 4°C for about a month. If stored frozen at -20°C or -70°C, it will remain stable for 2-3 months. Repeated freeze-thaw should be avoided, as it will denature the oligo. Avoid the use of distilled water, since solution pH may be as low as 4-5.
Does my oligo have a phosphate on the 5-Mod end?
Unless requested, oligos are synthesized without either 3-Mod or 5-Mod phosphate. The 5-Mod phosphate modification is available, normally as an additional charge.
My annealed oligos will not ligate. What is the problem?
Ligation reactions require a 5-Mod phosphate. If your oligos do not contain a 5-Mod phosphate, ligation will not occur. The problem can be addressed without ordering an additional oligo pair: phosphorylate your oligos enzymatically with kinase before use in ligation reactions.
How do you calculate the molecular weight of my oligo?
The molecular weights for oligos is the sum of the component molecular weights of all bases, with mixed bases contributing proportionately. The component molecular weights of the bases vary as to their salt form. Many times desalted oligos are ammonium salts, while cartridge, HPLC and PAGE purified oligos are sodium salts. The molecular weights used in the calculations are listed in the following Table III.
Where PA is the number of As and WA is the component weight of A and Pmod is the number of Modifications, and Wmod is the component weight of the added modification.
Please refer to Table IV for the molecular weights of common modifications. Table IV
Molecular Weights of Common Modifications
Modification
Molecular Weight
Modification
Molecular Weight
5-Mod-Biotin
405.45
3-Mod-TAMARA
623.60
5-Mod-(6 FAM)
537.46
3-Mod-Dabsyl
498.49
5-Mod-HEX
744.13
3-Mod-Fluorescein-dT
815.71
5-Mod-TET
675.24
3-Mod-(6 FAM)
569.46
5-Mod-Cy5
533.63
3-Mod-Amino Modifier C3
153.07
5-Mod-Cy3
507.59
3-Mod-Amino Modifier C7
209.18
5-Mod-Dabcyl
430.18
3-Mod-Thiol Modifier C3
154.12
What is coupling efficiency?
Coupling efficiency is a measure of the DNA synthesizers ability to couple each new monomer to the growing chain. If all the monomers coupled completely to the growing chain, the coupling efficiency would be 100%. If 1% of the growing monomer chain fails to react, then the coupling efficiency of that step is only 99%. The coupling efficiency for the complete synthesis of the oligo is usually determined from the yields of full-length sequence after the first and last cycle. Coupling efficiencies greater than 99.0% are essential for good oligo product with minimum purification.
How is the coupling efficiency determined?
Following the first coupling step, the amount of Trityl released during deblocking is directly proportional to the amount of full-length oligo made in the previous cycle. When the Trityl is cleaved during the deblocking step, the resulting Trityl cation is orange in color. The intensity of this color can be measure by UV spectrophometry. By comparing the intensities of the Trityl produced after the first and last coupling, one can calculate the average successful base coupling per cycle and hence the coupling efficiencies.
I sequenced a clone I prepared with your primer and the sequence for the primer region was different from the one I ordered. Why?
Base insertions are attributed to a small amount of detritylated amidite present during coupling, while deletions are probably due to failure sequences that don get capped and are subsequently extended.
However, a better explanation for the observation of altered sequences is the incomplete deprotection of the oligo. With a deprotecting group still on a few positions when the annealed and ligated oligos were transformed into E. coli, the host mismatch repair system would try to resolve these bumps with the results sometimes being the wrong base. The most likely culprit for incomplete deprotection is the isobutyryl protected dGs. These are the hardest deprotection groups to remove. If the oligos were vigorously deprotected a second time, mostly likely the new clones would have sequenced correctly. Also, in general, the longer the oligo, the greater the probability of side reactions accumulating along with increased chances of incomplete deprotection.
Why are some modified oligos so expensive in relation to the cost of the modifying reagent?
The limited reagent stability (most <48 hours) and lower coupling efficiencies of the reagent requires that excess modifying reagent be used to insure adequate quantities of full length product is made. As a result, higher cost incurred in synthesis.
Why are the yields lower for modified bases?
Many of the modified amidites are unstable and do not couple as efficiently as the unmodified bases (even though longer coupling procedures may be used), thus failure sequences are more abundant than in normal synthesis. Consequently, all modified oligos should be purified either by cartridge or HPLC to remove the more abundant failure sequences. Yields are reduced as a result of purification.. The end product, although with a lower yield, is much more pure.
Why is the yield for 1 µM scale syntheses not five times greater than 0.2 µM scale syntheses?
For 0.2 µM scale, the monomer coupling is done at a 40-50-fold excess. To do so for larger scale syntheses (such as 1.0 µM scale) would be cost-prohibitive. Large-scale syntheses are done at 10-fold mole excess of amidites. However, to increase the yields for these larger scale syntheses, the coupling times are extended to increase coupling efficiencies.
What is the longest length an oligo can be synthesized?
The real answers lies in the limit of resolution of the purification method and the coupling efficiency of the DNA synthesizer. It is not unusal to synthesize oligo in excess of 150 bases and to obtain sufficient quantities by PAGE purification to do successful gene construction. It should be remembered that the longer the oligo, the greater the chance of accumulated sequence errors.
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