I have an AAV plasmid and I need to insert a fragment that is 48 bp long. What's the most efficient way to generate the fragment? I have another plasmid that contains the fragment sequence. Can I PCR amplify the 48 bp portion that I need? Or should I order an oligo? If the oligo strategy, can I order 2 primers (cheaper than dsDNA oligo) and just anneal them?
Dear Eunice Lim
yes you can PCR amplify the target 48bp from the other plasmid and insert it into the AAV (transfer) plasmid backbone.
Note- Do not use the helper or the rep/cap AAV plasmid for this cloning step.
Also, you need to look/select for proper restriction sites to perform the entire cloning to insert the sequence into the AAV plasmid. After successful cloning make sure that you have correct inserts and stable ITR region by sanger sequencing and control digests.
Please read here for more details- https://www.azenta.com/blog/optimizing-aav-plasmid-preparation-and-itr-sequencing
All the best.
I recommend ordering two primers and annealing them in the annealing buffer rather than PCR. It is more reliable and faster to complete. However, you should check the sequence even if you are not using a PCR method.
Thank you Shambhabi and Yohei for your answers. I tried both methods in parallel (PCR amplification and annealing primers) to obtain the insert DNA. I found that the various constructs generated with the PCR amplified-fragment all sections of 10-20 bp missing. The constructs generated by annealing primers did not produce such a problem. I think PCR amplification may work but it requires extra steps of optimizing primers and annealing temperature. So I will probably opt for annealing primers to generate gene fragments in the future.
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