Currently doing basics of Molecular cell biology. And I have done a gel electrophoresis with DNA as part of RTPCR. Gel electrophoresis has mixes with DTT and one with Mg2+, which produced bands only for the reaction mix with DTT, followed by a restriction enzyme digest.
Im not quite sure how to verify if I have cloned the cDNA into the plasmid vector? How do i go about doing this.
Thanks!
That is simple: just run the plasmid vector alone in one lane and the vector with the cloned cDNA sample mixture in another lane and compare the band size.
You can't properly judge size in a circular plasmid on an agarose gel. You have to at least linearize for it to run true-to-size.
This is such a basic question that you need to either talk to your mentor or class instructor.
You could get the plasmid sequenced. A faster test would be to use PCR with 1 primer in the vector backbone and 1 primer in the cDNA insert. Your negative control in this case is the empty vector.
To assess whether you have successfully cloned the cDNA into the plasmid vector, you can perform several verification steps. Here are a few commonly used methods:
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
https://www.researchgate.net/post/Single_Huge_Horizontal_Line_Through_DNA_Gel_Electrophoresis
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