Hello Community!
I am sending some PCR product for Sanger sequencing, which requires me to send sequencing primers along with my samples. Would the sequencing primers just be the primers I used for PCR?
If not, how would I design a sequencing primer for my PCR fragments?
Thanks in advance!
https://dnacore.mgh.harvard.edu/new-cgi-bin/site/pages/sequencing_pages/primer_design.jsp
Check the link, please. You will get the criteria along with the software list. My personal preference is primer 3.
Yes, you can send the primers used for the PCR as sequencing primers and let the sequencing facility know about it.
One primer will be able to amplify around 1000bp of read and you might get 600-800bp read correct. Based on your insertion or whatever region you want to check you can use your existing ampifying primers or you can also check if it has any universal primers region inserted such as M13. Otherwise, you can design primers Forward amd reverse keeping in mind 800-1000bp amplifying region(each primer will cover 600-800 correct bp at least depending upon your DNA purity). You can design it in primer 3 or manually. Just check optimum primer conditions to amplify.
Yes, you can use same primers for sequencing PCR that you used for amplification of your target.
Same primers are used for Sanger sequencing which are used in PCR amplification.
Whether you can use your PCR primers as sequencing primers depends on what you did to purify your PCR product after amplification and what kinds of side-products were produced, even at very low levels, in that PCR reaction. The problem with using the same primers is that they will also work as sequencing primers on any of those other side products, like primer dimers etc. that are still present in the sample--and that may result in a garbled, unreadable sequence trace. If you gel-purified your PCR product, then the amplification primers might work for sequencing because you can be reasonably sure that the only template is the full length product. Other kinds of clean-up might also work, but the possible presence and amount of competing sequence template is what you should be concerned about.
You don't need to design new primers for sequencing. You have to send the same primer set (which is used in PCR amplification) along with the purified PCR products for sequencing.
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