6 replies to this topic

[HawkEye]

Hi!
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).

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[mdfenko]

why do you think it's not supercoiled? a supercoiled plasmid will run faster than a relaxed plasmid.

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.

[HawkEye]

So, i checked lower band by restriction mapping and PCR. it is not supercoiled form. any ideas what it can be? IMHO, it is some host plasmid of competent cells. how do U think, is it possible?

[pDNA]

if you suspect your competent cells to be contaminated then check them by doing a plasmid prep from your competent cells.
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!

Regards,
p

[HawkEye]

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.

[pDNA]

normally (most) of the companies know what they do and they have something like quality assurance and so on ...so i would more or less believe the problem is on your side

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?

Regards,
p

HawkEye, on 26 March 2011 - 05:32 AM, said:

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.

[HawkEye]

of course, it is more likely my fault    but the same problem appear in neighbor lab with same lot of cells. and as my colleages from this lab said today: when they tried another homemade competent cells - strange band disappeared. so, problem is in contamination of the commercial cells.