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cloning and activity problem

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cloning and activity problem

Postby denrul » Sun Oct 25, 2009 1:22 pm

Hi there;
I have two question about my cloning attempt,

1) I have cloned three different genes (originated from bacillus) in to LacZ of pSK(-) (pbluescript) vector. After transformation E.coli DH5alpha and induce with 0.5 mM IPTG there is no difference on SDS gel between Host and Clones. Cloned genes has own promotor region and Shine-dalgarno sequence. I have tried different IPTG concentration, different time of induction, different media but still I see same SDS gel profile. (Cloned vector has been checked for right orientation). What do you think about this situation. Unfortunately for my protein there is no antibody and I coudn't make western blot.

2) As you know pSK vector has LacZ promoter region and Shine-dalgarno sequence of B-galactosidase and Cloned genes has own promotor region and Shine-dalgarno sequence. Is this situation create any problem for activity.
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Postby JackBean » Sun Oct 25, 2009 1:32 pm

1) I did you sequence the insert after transformation?
II if zou have pBLUESCRIPT, zou should be able to perform white/blue test, shouldn't you?

2) sure, the cloned gene is driven by itselve promoter, so the IPTG can't induce expression
http://www.biolib.cz/en/main/

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Postby denrul » Sun Oct 25, 2009 2:16 pm

1) Yes I have sequenced my fragment and of course I selected my clone via white/blue screen.
2) When inducing with IPTG, Is quantity of mRNA increase?? Basically; increasing mRNA quantity cause increasing protein quantity so increasing activity of protein. I want to ask you is ribosome use SD sequence of B-gal ??
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Postby JackBean » Sun Oct 25, 2009 2:58 pm

It would increase, if it has been driven by the b-glu promotor, but if the gene has its own promoter, than probably not.
BTW how large is your insert? It must be really long, if it contains also the promotor.
http://www.biolib.cz/en/main/

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Postby denrul » Sun Oct 25, 2009 3:34 pm

inserts long 2 kb, 2.5 kb and 4,5 kb respectivelly.
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Postby JackBean » Mon Oct 26, 2009 2:26 am

Nice, but anyway, for IPTG induction, you must remove the natural promoter and keep the gene under b-glu promotor control ;)
http://www.biolib.cz/en/main/

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