6 replies to this topic

[Luria Bertani]

Hi there

I have discovered a box of One Shot TOP10 competent cells deep within our deep freezer. So I am very curious about how I can use this.
I have looked up a manual for these cells and it says that "TOP 10 Cells do not require IPTG to induce expression from the Lac promoter".

So my question is- if I have a plasmid from the pET system under T7 promoter control, I still need to add IPTG to induce my target protein right??

I am also looking at the genotype for this. What does Str^R mean? streptomycin resistance?

[Luria Bertani]

http://www.chem.eng....otTOP10_man.pdf was the link I got the info from

[protolder]

Luria Bertani, on 18 April 2011 - 12:26 AM, said:

http://www.chem.eng....otTOP10_man.pdf was the link I got the info from

Hola yes streptomycin resistance, but this is a subcloning strain that only need Xgal for blue/white selection, in fact after transformation it says check the insert presence by PCR or whatever.This is an efficient strain to transform with ligation mixtures equivalent to JM103or DH5 alpha as pDNA said to you in your question before. Buena suerte LB

Edited by protolder, 18 April 2011 - 02:40 AM.

[Luria Bertani]

Hi Protolder
Thank you for your reply. Has anyone had experience with using this particular strain for expression?
Would it be useful for that purpose, or it it more just a cloning strain for making plasmids?

[bob1]

Top10 are more designed for plasmid production rather than expression.  You would be better using BL21 or similar for protein expression.

[pDNA]

Please bear in mind that Top10 is not a DE3 lysogen ...therefore no T7-RNA polymerase on the genome and therefore zero expression using the T7 system!!!

Regards,
p

[Luria Bertani]

Oh OK, so that basically means that they won't work with most or all pET vectors then.
Thanks for this.

LB