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1. 10mM dGTP (can be any other dNTP)
2. T4 ligation buffer
3. NEBuffer 2.1 (NEBuffer 2 with BSA)¹
4. At least 18μl of spin column purified dsDNA
5. BSA ²
6. T4 DNA polymerase

Protocol

1. For each dsDNA species, mix 18μl of dsDNA with 2μl NEBuffer 2.1 in a fresh eppendorf tube.
2. Add 0.5µl T4 DNA polymerase
3. Incubate at 25°C for 15 minutes
4. 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
5. 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
6. Add 1µl T4 buffer
7. Incubate mix at 37°C for 30 minutes
8. Transform entire mix into competent cells - I usually transform into chemically competent cells

Footnotes

1. 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
2. NEBuffer 2.1 already contains BSA no additional one is needed