Perform Gateway cloning reaction. Add reagents in this order:
1 uL 100 ng/uL double Gateway plasmid
0.5 uL 50 ng/uL attL3-tRNA-gRNA-LeftHomologyArm-attL4 synthesized dsDNA
0.5 uL 50 ng/uL attL5-RightHomologyArm-attL6 synthesized dsDNA
0.25 uL LR Clonase II enzyme mix (Thermo Fisher)
Incubate at least 2 hours at 25°C in thermocycler with heated lid.
Important Note 1: Add the reagents in the order specified. DO NOT make a master mix of the double Gateway plasmid and the Clonase. Doing so will result in dramatically reduced cloning efficiency, often resulting in zero positive colonies. Apparently the Clonase needs to encounter the attL and attR sites together for efficient cloning.
Note 2: The published protocol specifies 1 hour incubation, but the double Gateway reaction is less efficient than single Gateway cloning and I have found that a longer incubation improves cloning efficiency. I often leave these reactions overnight.
Note 3: Thermo Fisher sells a Gateway Clonase that is advertised to be optimized for cloning multiple attL fragments into a single plasmid (as we are doing here). The enzyme is twice as expensive as the normal Clonase. So far, I have always had success with the original Clonase, so I haven't tried the optimized enzyme.
Note 4: Some users have reported that the mass of DNA provided by Twist can be off by as much as a factor of 3. They have found that measuring the actual concentration of the dissolved DNA and normalizing to 50 ng/uL substantially reduces the variability in their cloning efficiency.