I have cloned gene in one plasmid and sequence confirmed with Miniprep. I want to scale up same plasmid, for that I transformed my Final Miniprep plasmid into DH5 alpha cells and further used a single isolated colony for primary culture and same culture used to inoculate media for scale up. My scaled up plasmid is not showing band at same position of Miniprep plasmid on agarose gel. Sequencing results showing bad reads for scaled up plasmid. What are the probable reasons for scaled up plasmid not showing identity with original plasmid?
Mangesh,
How did you select for your plasmid when scaling up? Did you use the appropriate antibiotics? I've made a mistake in the past where I was working with many different kinds of vectors which required different antibiotics for selection, and while in a hurry, added the wrong antibiotic. This subsequently could lead to undesired bacterial growth, yielding a nonspecific band similar to what you described.
Mangesh,
David's question is good. I was also wondering if you have tried to miniprep from the putatively empty DH5 prior to transformation. In other words: are you sure that the hosting bacterial strain you have used was void of plasmid? I would run a control experiment. In facts, if you have another plasmid around, that could be the reason of your results.
Another question is: why did you apparently changed the hosting strain? From your description it seems that you have done cloning in a host and then moved to DH5. I have probably misunderstood. If not why not doing maxi-prep directly from the original transformant?
Hope I have been of some help.
Luca,
DH5 contains a recA mutation which prevents heterologous recombination, therefore increasing insert stability. That is probably why he selected the DH5 vector as opposed to his original plasmid harboring the insert. But anyways, Mangesh just downvotes peoples answers that try to help him out, so I'm not interested in helping someone who doesn't appreciate feedback from other people. Apparently he has all the answers, even though he is asking questions on this site and his experiment won't work.
Dear David,
Thank you for your answers. I have not downvoted anyones answers. Please check again.
Thank you Luca A Vitali. I have my cloned gene into Freedom™ pCHO 1.0 Expression Vector, transformed into DH5 alpha and sequence confirmed a colony which is used for Miniprep inoculation. I have used correct Kan antibiotic selection. Generally, I complete my cloning and perform Miniprep and store same Miniprep plasmid at -20. After few months depending on scale up requirements, i use same Miniprep palsmid for transformation into DH5 alpha for scale up purpose.
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