why competent cells take up only 1 type of plasmids? - (Sep/21/2006 )
I have a question. After ligation, two types of plasmids exist in the ligation mixture, i.e. re-ligated plasmid without the insert and plasmid ligated with the insert. During transformation of competent (made so with heat-shock method) E. coli (i'm using DH5-alpha) using this ligation mixture, the cells will only take up only one type of the two plasmids. Why is that? It would be great if anyone could provide me the relevent web page that discusses this phenomenon.
I'm using pQE30 by the way. I have picked 20 transformed colonies but none positive clone has been found, all have the re-ligated plasmid. Therefore, i have transformed a new batch of DH5-alpha and will screen them tomorrow. If i still don't get any positive clone tomorrow, i would suspect something is amiss with the ligation. However, i have no problem obtaining positive clones for another plasmid, i.e. pGex-4T-1, of which the experiment was carried out concurrently with the pQE30 plasmid. So have i left out some details specific to pQE30?
could you give us more details of your cloning protocol?
I would not assume that this is a problem with your comp cells, I would think it's much more likely to be a problem elsewhere in your protocol
i am assuming some problem with the cloning and not a problem of the cells.
If u r not digesting ur plasmids sufficiently, then u will only have religated vectors after transformation.
more details will help.
Well U'r not the only one with that problem which I believe is on the vector.
One of my collegues had the same problem which he decided to continue his work with the working vector. In his case 1/20 picked colonies had an insert and sometimes 0/20 while the other vector gave good % insert.
This is believed to the vector problem not your cells.
Thanks! But does anyone know the answer to my topic-titled question?
You will have only one of the two plasmids because one excludes the other (are from the same incompatibility group), and if You are not obtaining good cuts and religations, is more probable to get the plasmid without insert.
Hope it hepls,
i don't think it's a pb of exclusion. i think it's more because of :
1 the cell need to be open and the cell need to survive it
2 during the opening, a DNA should enter.
I think it's a rare event in regard of the numerous bacterias presentin the cuvette.
Moreover, the plasmid is destabilized with electricity isn't it?
i think the product coded by ur gene is toxic to the cell, so all positive clones must be dying off!!!
why dont u try something like a blue white selection, coz sometimes all 20 u picked by chance might really be negative. sometimes luck also plays a factor. just chance!!!!!