Hello,
I hope this message finds you well. I am currently planning to synthesize a long chain oligonucleotide using our oligonucleotide synthesizer. However, I find myself uncertain about the feasibility of this endeavor. The total length of the oligonucleotide is 256 base pairs.
My strategy involves synthesizing shorter fragments, each containing 20 to 22 base pairs, akin to primers. Subsequently, I intend to join these fragments using ligase enzyme.
I am reaching out to inquire if anyone within our community has experience with such a procedure. Specifically, I would greatly appreciate insights into the effectiveness of joining these fragments using ligase enzyme.
Thank you sincerely for any assistance you can provide.
Warm regards
Dr. Shahina Akter
Email: [email protected]
As far as the synthesis of your DNA fragment is concerned, I do not recommend making a synthetic 256-mer. On the other hand, making a set of 20- to 22-mers might be too conservative. You can choose a strategy in between these two extremes, for instance using a set of about 60- to 80-mer oligos. If your synthesizer works well, you should be able to obtain the required material in good yields and purity. If you decide to go this route, I recommend using solid supports, either nucleosidic or universal, based on CPG1000 for 60-mers and CPG1400 for 80-mers. You will have a lot better success in further steps of your project if you purify your crude oligos by either ion-pair reverse-phase HPLC, ion exchange HPLC, or gel electrophoresis. For relatively long oligos, the use of HPLC columns with the pore size of 100 A is recommended.
I have no first-hand experience in the ligation. I am sure someone will advise you on that.
Good luck with your project.
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