5 replies to this topic

[anonymous]

First of all, plate out your glycerol stock on an ampicillin selection plate and pick a few colonies and grow them overnight in 5ml of LB plus antibiotic. Then induce as normal. This way you can eliminate bugs that have lost the plasmid. Maybe this way you can get some expression.

[anonymous]

I am trying to express a soluble protein in BL21 cells with the pET-15b vector. To induce my protein, I use 1 mM IPTG for 3 hours at 37°C, but on Western Blot I don't see any difference in expression level between induced and uninduced cells. Can anybody help me?

[anonymous]

Hi,Sometimes these pET vectors leaks so you don't see a difference between the nduced and non induced. use as a control also  cells containing the plasmid w/o insert.2) there is now new bacteria from stratagene that suppose to control better the uninduced (prevent leaking ). 3)use less amount of IPTG. 4)do a timecourse expression experiment.good luck

[ragno]

bonjour,Je pense que vous devez comprendre le français, sinon faites le moi savoir OK !Tout d'abord, êtes vous sûr que le cadre de lecture du gene dans le plasmide est bon?Sinon vous pouvez essayer une induction avec différentes concentrations en iptg.vous pouvez essayer d'induire à 30°C.Avez vous essayé un western blotting? Parfois on ne voit rien en coomassie et c'est détectable en blot, cela m'est déjà arrivé.Bon courage.

[ragno]

Effectivement vous avez fait un blot alors peut être que l'Ac I est dégradé.Il faudrait mettre un contrôle positif pour vérifier votre AC I.

[anonymous]

I am having the same problem. However, I do not understand any FRENSH.  I think it is very unhelpful and ignorant to others who might have the same problem, not to response in ENGLISH!!