CLONING PROBLEMS-HELP

Sergio sergioal at NOSPAMbbm1.ucm.es
Wed Jul 19 13:05:45 EST 2000



"P. Moreno" wrote:

>  Dear colleges:
> I am making a contructor with pET vector; the question is:
> I ligate the vector with the insert and all looks nice, when I transform
> E.Coli and I make a mini-prep ( home mini-prep or kit mini-prep from
> quiagen ) the plasmid tha appear is littlest than the normal ( molecular
> mass 1Kb, when it should be of 6 Kb ). It don´t occurre all the time, at
> the first time was a 5%, but now is 60%.
>
> I had changed the bacteries and didn´t result.
>
> WHAT CAN I DO?

(Spanish version following.)

Hi,
probably the enzymes you are using to check the plasmid size aren't working.

I guess you're selecting the ampicillin resistant cells, so the bla gene
must be in the plasmid, as well as the ColE1 origin. However, 1kb is not
lenght enough for the bla and the ColE1, so that cannot be the linear
molecule (unless there are two bands of the same size). Usually a 6kb ccc
plasmid doesn't run so far (1 kb), what's the buffer and agarose percentage
in your gels?.
Have you performed a control to verify your competent cells are not
contaminated with another smaller plasmid?


Spanish version.....
Hola,
posiblemente las enzimas que estas utilizando para comprobar el tamaño no
estan cortando bien.
Supongo que estas seleccionando en ampicilina y por tanto el gen blsa ha de
estar presente en el plasmido. Sin embargo  1kb no es tamaño suficiente para
el gen bla y el ColE1, por tanto no puede tratarse de la forma lineal (salvo
que sea un doblete). Normalmente un plasmido de 6kb en forma ccc no migra
tan bajo, ¿que buffer y que porcentaje de agarosa estas utilizando?.
Has hecho algun tipo de control para confirmar que tus competentes no estan
contaminadas con otro plasmido mas pequeño?


Sergio








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