Extra band on gel after miniprep
Posted 24 March 2011 - 03:38 AM
I have a very strange problem. After miniprep i`ve got some extra band in my miniprep. And it is not supercoiled form of plasmid because it`s located very low (2.5-3 kB) according to my construct (13.5kB). Also I purified both bands and checked them by PCR and sequencing. As i expected - upper is my plasmid, lower - "something else". Also problem is that appropriate concentration of my plasmid was founded only in one miniprep from 8 (see attached picture).
As additional information: for transformation i used DH5alpha cells (not homemade).
Posted 24 March 2011 - 08:15 AM
why don't you try linearizing and running on the gel? then you will know if the lower band is supercoiled plasmid or something else.
genius does what it must
i do what i get paid to do
Posted 26 March 2011 - 12:50 AM
Posted 26 March 2011 - 03:06 AM
maybe the cells are not DH5a ...instead somebody inoculated a "natural isolate" ...this happens from time to time when people think they can skip the sterile control when preparing competent cells. Or do you bought the cells? ...then they should be alright and your source of contamination is elsewhere!
Posted 26 March 2011 - 05:32 AM
Posted 26 March 2011 - 05:53 AM
just linearize your plasmid and load it again ...as mdfenko suggested ...otherwise you don't know what you are looking at ...load undigested and linearized plasmid and we will see ...maybe you problems go away then.
maybe the plasmid prep you electroporated was contaminated with another plasmid? ...is that an option? is it possible to check the original plasmid once used to transform the cells?
thanks for answer. i also thinking about contamination but we bought these cells (i don`t want to write name of the company before we will find real reasons of problem). Anyway I will try to transform TOP10 cells with my plasmid, isolated from gel.
Posted 26 March 2011 - 10:38 AM