Will linearized plasmid still have drug resistance? - (Oct/06/2006 )
Hi, everyone,
I just have a stupid question, will the linearized plasmid still have drug resistance after introduction into competent cells? In another words, will this be a problem of false positiveness? A question has haunted me for a while...
Thanks!
no. Linear plasmid won't give rise to false positives.
(EDIT: Actually it can, but the number of false positives from this sourse is so low it can be ignored)
But be aware of the deantured plasmid, plasmid DNA that has been overly exposured to the alkaline lysis solution. Plasmid in this configuration will not be cut by restriction enzymes but will transform rather well. Thus always gel purify the plasmid vector before use.
But be aware of the deantured plasmid, plasmid DNA that has been overly exposured to the alkaline lysis solution. Plasmid in this configuration will not be cut by restriction enzymes but will transform rather well. Thus always gel purify the plasmid vector before use.
Oh my god, do you mean after miniperp, gel purification is needed before restiction digestion ?
Actually, I often find that the vector cannot be completely digested
no. Linear plasmid won't give rise to false positives.
But be aware of the deantured plasmid, plasmid DNA that has been overly exposured to the alkaline lysis solution. Plasmid in this configuration will not be cut by restriction enzymes but will transform rather well. Thus always gel purify the plasmid vector before use.
Oh my god, do you mean after miniperp, gel purification is needed before restiction digestion ?
Actually, I often find that the vector cannot be completely digested
Gel purify minipreps? Oh no no. There is no need to do any purification if all one wants to do is run a diagnostic digest. The denature DNA is only a small proportion of the total vector population. Usually undetectable visually in minipreps. Anyway it is best to keep the alkaline lysis time under 1 min, to prevent exessive build up of denatured plasmid.
This problem of denatured plasmid is mainly for transformation and ligation work, as competent cells are quite good at picking up plasmids (the smaller the better) and the original vector tends to be smaller.
However, gel purification of the restriction digest is a must...
as far as i know, the efficiency of transformation of linear plasmids is very low.
Thanks guys! Really appreciate your help!
as far as i know, the efficiency of transformation of linear plasmids is very low.
Thanks guys! Really appreciate your help!
But again, why linerized plamids have no drug resistance? why??
Thank you!
Most plasmids replicate only in circular form. A linear plasmid will typically be quickly degraded in the cell and fail to be replicated. While mRNA and protein may be produced by the plasmid DNA while in linear form, this is a very temporary situation, and will not persist as the cells replicate.
Thanks phage434, but again, why they only replicate in circular form?