I wonder if anyone could help me with a technical problem in this regard. I'm using the pJet1.2 plasmid (from CloneJet, Fermentas) to clone a 491 bp gene in E.coli DH5a. This bacteria holds no ampicillin resistance gene, so the growth observed in LB+amp plates is attributable to transformation with the desired plasmid+ insert. Insert size was previously checked in agarose gel. How can possibly be that the size of the plasmid I get after a miniprep is less than that of pJET 1.2 (2974 bp)??? The expected size is 3465 bp (plasmid + insert) but I got a band of 2,3 Kb using two different miniprep kits. I'll appreciate if someone can give me a clue.
i think thats normal, after a miniprep with commercial kits, you mostly get supercoiled plasmid that will travel faster and it looks smaller in size. sometimes, you might even see three bands, supercoiled, liner and nicked plasmid, depending on kit and handling.
To check the right size, do a restriction digestion with an enzyme that cuts once and you will get a linear plasmid, run it on gel and you will see the correct size on gel.
i think thats normal, after a miniprep with commercial kits, you mostly get supercoiled plasmid that will travel faster and it looks smaller in size. sometimes, you might even see three bands, supercoiled, liner and nicked plasmid, depending on kit and handling.
To check the right size, do a restriction digestion with an enzyme that cuts once and you will get a linear plasmid, run it on gel and you will see the correct size on gel.
Irfan is correct. Plasmids isolated from bacteria are supercoiled. Supercoiled DNA will run ~30% faster than linear DNA. 3465bp x 0.7 = 2425bp The band you are seeing is the expected size for your supercoiled DNA plasmid.
Congratulations on a successful purification!
Thanks Jason and Irfan! I came to that conclusion after checking all possible factors involved. So, I'll double check the size of the vector by restriction digestion and electrophoresis.
Once again, thanks for replying!
Hi Maria! another very fast thing you can do just to check if the insert is in the plasmid is to run an agarose gel with a mini prep of an empty vector (the one you used for cloning, not digested, just circular plasmid right after the miniprep) and in the next lane run the miniprep you made after the ligation, It's true that circular and supercoiled plasmids won't run according to their real "linear" size but in any case you should see that the product of your ligation runs slower, and its band should be at least a little higher on the gel than the empty vector. Good luck!
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