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Re: [ccp4bb] problem in transformation of pqe 30 clone |
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CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: problem in transformation of pqe 30 clone From: Cynthia Kinsland clk10 {- at -} CORNELL {- dot -} EDU Date: 2009-05-04 Using pQE30, any E. coli is an expression host. Because it uses the T5 promoter, you don't need an E. coli strain carrying the T7 RNA polymerase (so, you don't need a "DE3" strain). As noted by Artem, you are most likely having a leaky expression problem. However, it is odd that DH5a will transform if this is the case since it does not carry the lacIq mutation. XL1-Blue does, which is why it was suggested below. You could try expression right in DH5a, since you have the plasmid there. Your transformation difficulties seem strange. Using this vector, DH5a should not transform any more stably than the other strains you tried. I second the reclone it recommendation. Best, Cynthia On May 4, 2009, at 10:29 AM, Engin Ozkan wrote: > Have you tried M15[pREP4], which are the cells Qiagen would like you > to use? You can at least use pREP4 + your expression host, and have > repressor expression to prevent leaky expression. That can help you > get colonies of transformants. > > Engin > > > On 5/4/09 7:04 AM, atul kumar wrote: >> >> xl1-blue is not an expression host,since i have cloned it >> successfully,i need to transform into expression host, i am able to >> transform it into dh5 alfa,but not in any of expression host >> >> From: artem@xtals.org [mailto:artem@xtals.org] >> Sent: Mon 5/4/2009 6:32 PM >> To: atul kumar >> Cc: ccp4bb@jiscmail.ac.uk >> Subject: Re: [ccp4bb] problem in transformation of pqe 30 clone >> >> Hi, >> >> You are using a pQE vector which has a T5 promoter. T5 is a native- >> like >> promoter, recognized by the E. coli RNA polymerase - and this means >> that >> even with lac operatorsupstream there is a huge amount of leakage >> in this >> system. If your protein is even moderately toxic then you have >> issues. >> >> Your solutions are >> 1) to use cells with higher levels of lac repressor (XL1-blue for >> example) >> 2) to re-clone this ORF under some tightly controlled promoter >> >> Artem >> >> > >> > i have cloned my gene successfully into qiagen vector into pqe30 >> but i do >> > transformation of this into BL21,pLys,Rosseta,C41, i dont get any >> > colonies,comp cells are good other clones give good no of >> colonies upon >> > transformation. >> > can someone help??? >> > thanks >> > atul >> > CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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