I am new to plasmid DNA transformation and the steps that entail, so I would like to clarify i the steps I am about to take are correct:
1. I have received my gene of interest cloned in a pcDNA3.1 vector (from addgene). I will proceed to streak the bacteria onto agar plates and after 12-16 hours, pick single colonies and inoculate into liquid LB broth tubes. This will be left shaking at 37 deg overnight.
2. The following day, I will proceed to crate glycerol stocks of some colonies, and for some of them, perform plasmid mini prep to extract the plasmid DNA from the e.coli.
3. I would like to verify that my plasmid indeed has the gene of interest, and for that, I would like to first perform restriction digest. Here, I have a doubt: Do I need to amplify my plasmid DNA via PCR before restriction digest? Or should I linearise the plasmid DNA-->PCR-->restriction double digest to isolate my gene of interest -->run gel ? This part I am confused, as many sources say that PCR should be done prior to do restriction digestion verification. However, I do not read anywhere about linearising the plasmid DNA. Can I just amplify the circular DNA without linear zing?
Thank you very much and please feel free to comment if I have missed out any important steps!
Best Regards,
Mathangi
You do not need to PCR first for a restriction digest of a miniprep. Just digest the a small amount of the mini prep 200ng-1ug with restriction enzyme. Preferably with ones that cut in your insert. If you require additional verification you can sequences the insert from the miniprep.
Hanna Alalam While at it, may I clarify with you as to why in this addgene page, PCR is asked to be done first before restriction digest in plasmid cloning? https://www.addgene.org/protocols/pcr-cloning/
The page you sent describes the procedure for cloning into a plasmid since you already got the finished plasmids the only thing you care about is the last two subheadings in the page.
My process is a little different from yours. I don't extract the plasmid immediately after growing the colony, it's a waste of chemicals and time if it doesn't carry the gene. I do the PCR reaction first. If it has a target gene, I will extract the plasmid and take the next steps.
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