I am doing my final year project on amplifying variable heavy and light chains, the gene of interest was inserted in pEt16-iGg, and has been extracted and purified. The expected size of the genes are 300-400 bps, which is clearly not reflected on the gel. My supervisor is saying to indicate which is the plasmid and the PCR products, but I can’t find anything online. I hope someone can help. Thank you.
Your supervisor is asking you to distinguish between the plasmid and PCR products on the gel. One way to do this is to run a lane of the plasmid DNA alone and compare it to a lane containing the PCR products. The plasmid DNA will appear as a single band corresponding to its size, whereas the PCR products should appear as multiple bands, representing different sizes of amplified fragments.
Alternatively, you could consider using a gel extraction kit to purify the PCR products and then re-run them on a gel to see if you get a clearer picture of the expected size range.
It's also possible that the unexpected band sizes could be due to issues with the PCR reaction, such as non-specific amplification or incomplete amplification. It might be worth troubleshooting the PCR protocol to see if you can optimize the reaction and improve the band sizes.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
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