4 replies to this topic

[arvinsign]

Hi..i would just like to ask for comments. Im planning to clone a whole gene in a pBAD plasmid. In this case i dont want to use the pBAD promoter, but instead the native promoter of my gene, so in short its a promoter + ORF. Im having problems with finding good restriction enzymes sites in other plasmids and i i like the MCS of the pBAD. So i want to clone a whole gene downstream of the pBAD promoter (hoping that the presence of the pBAD promoter upstream will not in any way affect the expression of my gene). Do you think this will affect the expression of my gene? Thanks

The upstream sequence of my gene will be around 250 bp from the native +1 site.

[fishdoc]

arvinsign, on Aug 9 2009, 09:18 PM, said:

Hi..i would just like to ask for comments. Im planning to clone a whole gene in a pBAD plasmid. In this case i dont want to use the pBAD promoter, but instead the native promoter of my gene, so in short its a promoter + ORF. Im having problems with finding good restriction enzymes sites in other plasmids and i i like the MCS of the pBAD. So i want to clone a whole gene downstream of the pBAD promoter (hoping that the presence of the pBAD promoter upstream will not in any way affect the expression of my gene). Do you think this will affect the expression of my gene? Thanks

The upstream sequence of my gene will be around 250 bp from the native +1 site.

Insert your gene in the opposite direction as you would if you were cloning off the pBAD promoter. Alternatively, you could put a transcriptional termination sequence upstream of your promoter so that the pBAD promoter doesn't read through to your gene.

[arvinsign]

thanks fishdoc. i'll insert it in the opposite direction. I'll let you know the results later

[phage434]

You should not be limited in your thinking about the sites available in a particular MCS.  It is very easy to design primers that will amplify a vector containing any restriction sites you'd like to see.  Just remember to put sufficient 5' overhangs to eliminate cutting problems, then treat the vector PCR just as you would an insert PCR reaction.

[perneseblue]

as an addition to phage434 suggestion. As you can PCR amplify the vector to add any restriction site that you desire, you can also eliminate the pBAD promoter that you do not want to use. That would eliminate the double promoter problem.