The Milan protocol ...

SLIC Sub-cloning using T4 DNA polymerase treated insert

• Digest 3 μg vector with two restriction enzymes. Gel-purify the vector and isolate the DNA using Qiagen extraction kit. Quantify the vector by spectrophotometric reading.

• Amplify inserts using a proof-reading polymerase. Set up a 100 μl PCR reaction with 200 μM of each dNTP, 1 μM of each primer. Cycle as follows: 94°C for 5 minutes; 30 cycles of 94°C for 45 seconds, 54°C for 45 seconds, and 72°C for 1 minute (according to insert size); 72°C for 10 minutes. Gel-purify the inserts and isolate the DNA using Qiagen extraction kit. Quantify using a spectrophotometric reading.

• Take 3 μg of the vector and 3 μg of the insert and treat separately with 0.5 U of T4 DNA polymerase (1 μl) in T4 buffer (buffer 2 NEB) plus BSA in a 60 μl reaction at room temperature for 30 minutes. Stop the reaction by adding 1/10 volume of 10 mM dCTP and leave on ice. Scale reagents according to your needs.

• Set up a 20 μl annealing reaction using 1:1 insert to vector ratio with 500 ng of a (5 kb) vector, 1x T4 DNA ligation buffer (NEB), appropriate amount of insert, and water. Incubate at room temperature for 30 minutes. Leave on ice or store in -20oC.

• Add 10 μl of the annealed mixture to 100 μl of TOP10 chemical competent cells, incubate on ice for 30 minutes, heat shock at 42°C for 45 seconds, return to ice for 2 minutes, add 0,9 ml of LB, recover at 37°C for 1 hour.

• Plate onto Amp or Kan plate; incubate at 37°C overnight.

This protocol worked with the following reagents:

• vector = pGEX-2rbs opened BamHI/SalI
• insert = 0,9 kb fragment
• oligos = SL20/ SL30/ SL40 with 20,30,40 nucleotides annealing ends respectively.

Under these conditions, oligos with 40 nucleotides overhangs yielded 80% of inserted fragment regardless of the vector to insert ratio, whilst only 15-20% of success was reported with the 20 nucleotides overhangs oligos.