Hello, does anyone have experience with single-step BP-LR Gateway cloning? I'm having problem to clone a 3.2 kb fragment. I did the same gateway cloning for an 800 bp fragment and I just saw this morning that some colonies grew that may carry the 800 bp clone. Both fragments are regulatory elements (promoters) and not coding sequence. The 800 bp is 35S promoter and the 3.2 kb is an endogenous promoter in tomato. The 35S is a PCR-amplified fragment whereas the 3.2 kb fragment is a synthesised DNA that is already cloned in a small vector. So, I cut/linearised the vector carrying the 3.2 kb prior to gateway reaction. Both have the correct attB sequences. The destination vector is pHGWFS7,0 (13 kb). The donor vector is pDONR221. I attached reference protocols for single-step BP/LR gateway. Any comment and advice will be really appreciated, thank you
Hi Prashant, thank you for your input. I incubated the reaction for 16 hrs. I also have done longer incubation time (24 hrs) but still no results. I am aware that one-step BP/LR reaction is less efficient than the common two-step gateway though.
What vector is your 3.2kb promoter is cloned into? Are you failing to get any colonies after transformation of your Gateway reaction with the 3.2kb sequence, or do you get colonies that have the wrong plasmid?
Hi Daniel, the 3.2 kb promoter is cloned into pUC57-Kan. It is a synthesised DNA and I included the attB sites flanking the promoter. I did not get any colonies in this gateway reaction. I linearised the pUC57-Kan-3.2 kb prior to gateway.
The second reaction I want to clone 35S (800 bp). The promoter was PCR-amplified. I got some colonies from this reaction but have the wrong plasmid.
Hi Cahya. Isolating the wrong plasmid suggests that you have another AmpR plasmid in your mixture, for example a template plasmid used for PCR of the 35S promoter. Try gel isolating your PCR product first. It would also be helpful to obtain a positive control for the Gateway reaction; a potential concern is that the recombinase has lost activity and is the cause of the failed cloning. In addition, you may want to check the competency level of your bacterai- low transformation efficiency could also be a factor here.
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