1. ALL DEST and DONR vectors MUST be grown in ccdB tolerant cells. These can be obtained from Invitrogen, along with the BP and LR clonase. You must ALWAYS grow them with chloramphenicol selection (along with other appropriate selection - amp or kan).
2. The available ccdB tolerant strains can be made electrocompetent with standard protocols.
3. The attP4 and attP1R sites (and their derivatives) can undergo self-recombination. If you are using plasmids with these sites (e.g. pDONRP4-P1R), there is a standard protocol to follow to select for preps that do not undergo self-recombination. Click on the following (.pdf file) to see the protocol. This is based on a protocol from Marian Walhout's lab here at UMass Med.
4. The other DONR plasmid (pDONR221) and Dest vectors usually behave well. That being said, we always do a test BP or LR reaction on new preps just to make sure.
5. For multisite LR reactions, LR clonase Plus must be used. LR clonase II will not work. Conversely, LR clonase Plus will not work for standard LR reactions
6. It is possible to generate single fragment Destination vectors from any construct derived from a multisite cloning reaction. For example, we use two-way multisite to generate promoter:reporter constructs (attB4-promoter-attB1-egfp-attB2). To replace the promoter fragment, add the B4/B1/B2 plasmid with pDONRP4-P1r and perform a standard BP reaction. Instead of introducing the reaction into standard bacteria, transform ccdB tolerant cells, then select on chloramphenicol. The resulting plasmid will now have a Destination cassette (flanked by attR4-attR1) upstream of the egfp reporter. This plasmid will allow single transfer of promoter fragments from attL4/attL1R flanked ENTRY plasmids.
7. For more general details (i.e. not immediately related to zebrafish) on using the Gateway cloning system, go to Invitrogen's website.