My first post here on this forum. Been BP cloning all of last year a number of different PCR products with the gateway system with no problems but suddenly everything stopped working. I already got new enzyme, use newly extracted plasmid pDONR201, and check my PCR products for the correct sequence. In the past I normally didn't try the positive control but multiple tries resulted in failure to get bacterial colonies. Are there any ways to troubleshoot this problem?
My current strategy was to BP Clonase a PCR product into an entry vector (pDONR201) then LR clonase the entry vector into pDEST15 for bacterial expression. Would switching to TOPO TA cloning be a viable option to replace the BP clonase? Any help would be greatly be appreciated. Been banging my head at his for 8 months now with no luck.
Entry vector plasmid: pDON201
Destination vector plasmid: pDEST15 for bacterial, and pEGHA for yeast
PCR products contain the gateway sequence designed into the corresponding primers.
Use 1 ul of the BP clonase reaction to transform 50 ul of the competent cells.
Heat shock for 30 secs --> 42 C then 2 mins on ice --> Add 400 ul of SOC media and shake at 37 for an hour --> lightly spin down tube to concentrate cells and plate onto Kanamycin plate --> 37 C overnight