I am currently trying to Gateway clone a large PCR fragment. I have been able to PCR it perfectly and purify it through gel extraction.
However, after BP reaction and transformation I have no colonies (sometimes 1 that does not contain the insert).
I let the BP reaction incubate for 15 hours and use TOP 10 chemically competent E.Coli. (heatshock at 42°C for 30 seconds and plating everything).
Does anyone have any tips?
Hi Jeroen,
Do you precisely adjust the concentration of the DNAs in the reaction?
I believe that the purity, concentration and molecular ratio of the DNAs are most important in BP reaction.
Thanks.
Hi Takefumi-san,
I tried to adjust the values so that there was about an equal amount of molecules in the reaction. The ratio was about 1,1 plasmid molecule for every 1 PCR molecule.
To be honest, 10 kb is rather large for BP cloning, but not too large. Please try very high competency bacteria such as Mach1. Don't forget to incubate the cells at 37°C for an hour before plating after adding SOC.
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