I am presently using the gateway cloning system. Having managed to generate my own gateway compatible vector, l want to make my final construct. BP rxn was perfect. However, LR rxn is not working. From their protocol, you presently do not need to linearize the pDONR or vector. However, in the protocol to generate the vector, they mentioned linearizing the vector or if no suitable RE are present, to use topoisomerases. Does anyone have an experience with or without linearizing especially in a converted vector?
Daniel- I routinely perform Gateway recombination without linearizing any of the vector (all are circular, supercoiled). Can you provide some more details of the protocol you are using, and the bacterial host strains?
Daniel, l am just following the gateway protocol. Using 150ng of my vector and same amount of purified BP reaction into Omni Max cells by transforming 1 ul of a 10 ul rxn and spreading on ampicillin plates for selection . I screen with primers that comes from my vector across my GOI. I only get very faint bands at a specific point which does not correspond to the size of any of my GOI.
Daniel- what is the pENTR vector that you are using? Do you have another pENTR vector you can use as a positive control to check the activity of your LR clonase? Have you sequenced your Gateway compatible vector to make sure it has all the correct sequences?
l am using pDONR/ZEO and used the Rfa for the ccd insertion. l have used the vector for several cloning work and have sequenced it every time l manipulate it. It is working fine. l have ordered new clonase anyway. l guess putting it in a invitrogen made vector might be easy and not require linearization. Dont know about generated vectors. what do you think?
I generated my own Gateway compatible vector and it worked fine. Problems with improperly stored or old Clonase will do you in every time, though.
Can you post some maps of your two plasmids (even hand-drawn would be helpful)?
Hi Daniel. Is this your entry vector or your destination vector? I could not find the att recombination sequences in this plasmid sequence. Where are they located?
This is the destination vector. The entry vector was a pDONR/ZEO from invitrogen which l used for the BP reaction. See the plasmid which l annotated from the invitrogen site.
This destination vector should have the ccdB cassette, right? Otherwise you cannot select for recombination. Where is the ccdB cassette in the pLEW BSD plasmid?
The ccdB cassette will replace the luciferase cassette in the destination vector. goes into the HindIII/BamHi region. l checked by digestion and also transformation on the ccdB cells compared with Omni max cells. No growth on Omni max but several colonies as expected on ccdB e coli compatible cells
Oh- I thought you had post the map of the final destination vector. So you took the pLEW BSD plasmid, removed the luciferase gene, and inserted the ccdB cassette? Does this cassette include the flanking attR sequences?
l think so. From their protocol it is quite simple. Just blunt the ends if required, then ligate on the ccdB gene which has the compatible attR's with the BP clones. Hence when you perform the LR, it should be easy and simple. The pLew BSD is the destination vector and the attR's are the usual 25 nucleotides from invitrogen and gene specific nucleotides about 18.
Ok. Hope the new lot of Clonase solves the problems. Please post an update when you retry the recombination with the freshly acquired enzyme!
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