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: 94oC for 5 minutes; 30 cycles of 94oC for 45 seconds, 54oC for 45 seconds, and 72oC for 1 minute (according to insert size); 72oC 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 42oC for 45 seconds, return to ice for 2 minutes, add 0,9 ml of LB, recover at 37oC for 1 hour.

• Plate onto Amp or Kan plate; incubate at 37oC 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.