Problem: Our lab has a DNA synthesizer and sometimes I make in-home synthesized oligonucleotides.
From a certain time I have a problem with synthesis of long (>40 nt) oligonucleotides.
Short oligos are produced, but synthesis efficiency wanes rapidly with increasing oligo length, as I can judge from observing de-tritylation steps.
I already changed reagents, except the activator solution, which is 0,25M ethylthio tetrazole in catonitrile (ETT solution). I cannot immidiately replace the activator solution because I have no more the ETT stock.
My question:
Can I add phosphorous penthoxide (P2O5) directly to the activatoir solution in an attempt to dry out possible moisture? If this result in the appearance of (supposedly small) amounts of phosphoric acid - will this kill the process of oligosynthesis?
Or may be not?
To my knowledge, P2O5 is insoluble in acetonitrile, but I am concerned with the H3PO4 build-up.
Molecular seiver is used for drying reagents for DNA synthesis, in usual.
I do add molecular sieves 3A to the activator solution. In this case does not help, I do not know why.
In relation to my previous question: would anyone kindly explain,
to my understanding, the reason for which the activator must be prevented from moisture accumulation is not the ETT hydrolysis, but inefficiency of synthesis in the presence of water.
To my knowledge, ETT itself is stable in the presence of water. But one needs to prevent ETT to accumulate moisture and to dry crystalline ETT over drying agents so that no trace water kills the synthesis.
Is this right? ETT itself does not degrade if contacts water?
No, you absolutely cannot add P2O5 to the bottle with the activator. Your observations that shorter oligos are made efficiently, while longer ones are not begs a question: what is the pore size of your CPG or PS. You should conduct the synthesis of >40-mers using CPG1000. The wide porous PS is available, for instance, from Glen Research.
Thank you for your reply. I rouninely use CPG1000, always for long oligos (>50 nt), and sometimes even for short oligos because we do not need large amounts of oligos. Rather, we need the long oligos.
It seems, I figured out why long synthesis failed. I changed all reagents and prepared all solutions under argon and it works fine now for 60-mers.
Hi, Alexandr V Shustov ...
I believe you indeed figured it out: the reagents must be really fresh, especially the amidites. I work at Exxtend, an oligo synthesis enterprise in Brazil, and there we never use amidites more than one day old. They are always prepared and used in the same day. Moisture in the reagents and the absence of argon to prepare reagents do harm the synthesis efficiency but to my experience, the amidites are the most critical point here.
Thank you, Thaíssa!
Exactly, I finally figured out that amidites (undissolved powder) can be handled without overly complex precautions such as under argon, but before you prepare a solution you need to dry them under vacuum.
I weight needed amounts of amidites, put into glass vials with some small addition of activated molecular sieves and put the open vials under vacuum (oil vacuum pump,
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