expression vectors - expressing inserts in either orientation (Oct/08/2006 )
I want to randomly clone amplified PCR products into a vector, but I want the insert to be expressed. Therefore, I need a promoter on either side of the insert so irrespective of which orientation the insert goes in, I still get expression. Does anyone know of a vector that can be purchased that fits this criteria? Also, if the promoters are inducible by arabinose or IPTG, that would be a bonus. Thanks.
-ML1975-
I don't know of one off hand, but what is wrong with half of your clones expressing? Colony PCR can tell you what the orientation is, if you need to know, by using one primer on the insert and another on the vector.
-phage434-
QUOTE (ML1975 @ Oct 9 2006, 01:48 AM)
I want to randomly clone amplified PCR products into a vector, but I want the insert to be expressed. Therefore, I need a promoter on either side of the insert so irrespective of which orientation the insert goes in, I still get expression. Does anyone know of a vector that can be purchased that fits this criteria? Also, if the promoters are inducible by arabinose or IPTG, that would be a bonus. Thanks.
if I under stand correct you want a vector with two promoters?
promoter 1 - insert - promoter 2
Such a construct can be made but it will produce problems for the gene expression. mRNA silencing and transcription problems. (if strongly expressed). As suggested by phage434, colony PCR would solve the orientation problem quickly.
-perneseblue-