pEt 28-a expression system - (Apr/14/2006 )
Dear all
I have tried to clone a gene 2.3 kb in pET-28-a but I as not successful. I see more than one band in gell analysis after digestion of pET with two enzymes. Would you kindly tell me that is it possible that the band be related to the pLysS that is in BL21?
What points should I notice to clone this long gene in this vector to gain clones with my gene inserted in pET?
I would appreciate if you kindly help me.
Nozhat 
would you please explain a bit more clearly what you have done?
have you transformed into BL21 to get your clone? I find that an initial transformation into a more friendly strain is good (JM109, DH5alpha, etc) then move to BL21 after you have gotten the clone...perhaps it is just me but I usually get much higher efficiency that way
so I am not sure what you are seeing vs what you are supposed to see...could you explain a little more please?
have you transformed into BL21 to get your clone? I find that an initial transformation into a more friendly strain is good (JM109, DH5alpha, etc) then move to BL21 after you have gotten the clone...perhaps it is just me but I usually get much higher efficiency that way
so I am not sure what you are seeing vs what you are supposed to see...could you explain a little more please?
Dear aimikins
Thank you for your reply. yes I have transformed it in BL-21 first but I realized that it is not efficient. After digestion of pET 28-a , which in preformend from BL-21 host, with Sal I and EcoRI I see more than one bands. My answer is that Is it possible that the bonds are related to pLysS or not?