...
- 10mM dGTP (can be any other dNTP)
- T4 ligation buffer
- NEBuffer 2.1 (NEBuffer 2 with BSA)1
- At least 18μl of spin column purified dsDNA
- BSA
- T4 DNA polymerase
...
- For each dsDNA species, mix 18μl of dsDNA with 2μl NEBuffer 2.1 in a fresh eppendorf tube.
- Add 0.5µl T4 DNA polymerase
- Incubate at 25°C for 15 minutes
- Add 2µl of dGTP to the reaction, place on ice
Note: dsDNA treated with T4 DNA polymerase can be stored at -20°C for about a month - Mix equimolar amounts of treated dsDNA in a final reaction volume of 9µl
Note: I usually assume each sample is at roughly the same mass concentration and scale amounts - Add 1µl T4 buffer
- Incubate mix at 37°C for 30 minutes
- Transform entire mix into competent cells - I usually transform into chemically competent cells
Footnotes
- Though NEB says that T4 DNA polymerase retains full activity in CutSmart buffer, I believe this references the polymerase activity, not the exonuclease activity. SLIC did not work well with CutSmart buffer, but works very well with Buffer 2.1 -AJH