1. Digest 2 μg of vector with restriction enzymes. Gel purify the vector and isolate the DNA using QIAEX II gel extraction kit. Quantitate the vector.
2. Amplify inserts using Taq DNA polymerase. Set up a 100 μl PCR reaction with 250 μM of each dNTP, 0.5 μM of each primer, and 2.5 U of Taq DNA polymerase (from Eppendorf). Cycle as follows: 94°C for 45 seconds; 30 cycles of 94°C for 45 seconds, 54°C for 45 seconds, and 72°C for 1 minute; 72°C for 10 minutes. Add 20 U of DpnI to 100 μl of PCR products after PCR, incubate at 37°C for 1 hour (not necessary if going from a MAGIC vector to ColE1 origin). Purify the PCR products by QIAquick PCR purification column. Quantitate the inserts. We typically have 20 bp homology between the vector and the inserts.
3. Take 1 μg of the vector and 1 μg of the inserts and treat separately with 0.5 U of T4 DNA polymerase in T4 buffer (NEB) plus BSA in a 20 μl reaction at room temperature for 30 minutes. Stop the reaction by adding 1/10 volume of 10 mM dCTP and leave on ice.
4. Set up a 10 μl annealing reaction using 1:1 insert to vector ratio with 3 ng or less of a 3.1 kb vector (0.0015 pmol), 1x ligation buffer (NEB), appropriate amount of insert, 20 ng of RecA protein (Epicentre Biotechnologies) and water. Incubate at 37°C for 30 minutes. Leave on ice or store in -20°C.
5. Add 5 μl of the annealed mixture into 150 μl of BW23474 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 SOC and recover at 37°C for 1 hour.
6. Plate 100 μl onto plates containing the appropriate antibiotics and incubate at 37°C overnight. (Cl-Phe is used for vectors that have PheS-G294 between restriction enzyme sites. We have found that most background comes from uncut vector in our preps and therefore we can select against it with Cl-Phe. However, Cl-Phe is not an essential step and usually only has a 2-fold effect on background.)