cloning problems in C. elegans expression plasmid - Trying to use Fire vector pPD49.83 (Nov/13/2009 )
I am using the plasmid pPD49.83 from Andrew Fire kit. It has a heat shock
promoter in it and I am trying to put cDNA downstream of the promoter.
Also, I want to replace the promoter with my own promoters. But my
clonings are just not working. So, I wondered if you have faced similar
problems with using pPD vectors before. I am using NheI/SacI or NheI/NcoI
or SphI/BamHI for different clonings. Do you think some enzyme
combinations don't work well together?
Also, I was wondering if there is a Gateway system for C elegans
expression. Do you know about this? Are there any alternative/ easier
cloning systems like Gateway for C elegans?
Whats your final goal? Do you want to drive expression of your cDNA using heat-shock promoter or your promoter?
I am only concerened about star activity of BamHI, otherwise I don't see the problem. I agree they are not the greates quality vectors but noticed that worm people are really fond of them.