- 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.
- 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.
- 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.
- Set up a 10 μl annealing reaction using 1:1 insert to vector ratio with 150 ng of a 3.1 kb vector (0.074 pmol), 1x ligation buffer (NEB), appropriate amount of insert, and water. Incubate in 37°C for 30 minutes. Leave on ice or store in -20°C.
- 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.
- Plate 100 μl onto Cl-Phe/Kan 50 μg/ml plate; incubate in 37°C for 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.)
For Cl-Phe/antibiotic plates: 0.5% yeast extract, 1% NaCl, 0.4% glycerol, 15 mM DL-p-chlorophenylalanine (Sigma C6506), 2% agar. DL-p-chlorophenylalanine will not dissolve until after autoclaving. Glycerol and antibiotics are added after autoclaving. (We use a higher Cl-Phe concentration for this vector than in normal MAGIC recipients because the PheS gene in pMAGIC is expressed at a much lower level than in normal MAGIC recipients and therefore requires a higher Cl-Phe concentration.)
For SOC: 2% tryptone, 0.5% yeast extract, 0.05% NaCl, 2.5 mM KCl, 10 mM MgCl2, and 20 mM glucose.