What is the effect of particulates on oligonucleotide synthesis?
The existence of particulates in oligonucleotide reagents such as diluents, deblocking solutions, activators, capping reagents, and oxidizers can cause the clogging of the oligonucleotide synthesizer lines. The clogging of any individual line would of course be highly detrimental to the synthesis. For instance, if the deblock line is fully clogged, the deblock will not reach the column, and the synthesis will not proceed any further.
To prevent particulate contamination, we filter all our reagents through 0.2 µM filters. We also perform a visual examination to confirm that all our reagents are clear and free from particulate contamination.
Products:
Ancillary Reagents
How do I determine reagent consumption?
You have likely seen Coupling Data tables on our product pages and quality documents that describe roughly how many additions one can achieve using a certain pack size on various synthesis scales for ABI and Expedite instruments. These tables account for material required for sufficient priming. If you are looking to estimate the amount of material you need for a synthesis for parameters outside of those tables, here are some tips:
- At 0.1M amidite, one coupling that uses about 100 µL consumes roughly 10 mg (10 µmol) amidite. This approximation is based on a 1 µmol scale synthesis and assumes the amidite has a molecular weight of ~1000 g/mol. In this case, 10 mg comes out to 10 equivalents.
- Amidite consumption will also depend on the scale you are working on. For any given instrument, amidite usage is less efficient at smaller scales (Table 1). These are rough numbers and are meant to represent a good place to start when trying to estimate how much material you need for your synthesis.
- Liquid reagent consumption is largely dictated by the synthesizer. See below for some estimates on reagent consumption for two different instruments that operate on different scales (Table 2).
- Please keep in mind that reagent consumption is ultimately determined by the synthesizer. When in doubt, consider reaching out to your synthesizer manufacturer for a clear estimate of how much reagent is needed for your synthesis.
|
Scale (µmol)
|
Amidite Consumption (mg)
|
|
1.0
|
10
|
|
0.2
|
7.5
|
|
0.05
|
5
|
|
|
Reagent
|
ABI
(1.0 µmol)
|
ABI
0.2 µmol)
|
Dr. Oligo
(0.2 µmol)
|
|
Amidite
|
150 µL
|
110 µL
|
100 µL
|
|
Deblock
|
1.86 mL
|
1.32 mL
|
400 µL
|
|
Activator
|
370 µL
|
400 µL
|
200 µL
|
|
Cap A
|
250 µL
|
290 µL
|
100 µL
|
|
Cap B
|
230 µL
|
250 µL
|
100 µL
|
|
Oxidizer
|
420 µL
|
410 µL
|
150 µL
|
|
|
