Molecular cloning 81

Restriction enzyme digestion of DNA was performed as per manufacturer’s specifications (New England BioLabs Inc, Ipswich, MA, USA or Roche Diagnostics, Basel, Switzerland). In brief, for the specific restriction enzymes used within this study, a digestion reaction containing target DNA, 1 U enzyme µg-1 _{DNA, 1× enzyme-specific}

buffer and if required, 1× bovine serum albumin (BSA) (New England BioLabs Inc, Ipswich, MA, USA), was incubated for 1-2 h in a 37°C water bath. Double digestion completion was compared to single digestion controls by visualization on a 1% agarose gel as above and subsequently purified with QIAquick PCR Purification kit (Qiagen, Venlo, Limburg, Netherlands) as per manufacturer’s protocol with final elution in 10 mM Tris·Cl, pH 8.5.

2.10.2 DNA ligations

Ligations of DNA were performed for 1 h in a water bath at 16°C with reactions containing 40 U T4 DNA Ligase (New England BioLabs Inc, Ipswich, MA, USA), 1× T4 Ligase Buffer (New England BioLabs Inc, Ipswich, MA, USA), and appropriate concentrations of digested vector and insert. A standard reaction had ~100 ng total vector DNA and ~1 µg total insert DNA, calculated from a molar ratio of either 1:1 or 3:1 (insert:vector), based on the size variation of the insert compared to the vector. Blunt-end ligations used a molar ratio of 5:1 (insert:vector).

2.10.3 Competent cell preparation

2.10.3.1 Chemical competent E. coli preparation (rubidium chloride; RbCl)

E. coli strains were inoculated 1:100 from stationary phase (O/N growth at 37°C) into

PSI broth and grown aerobically at 37°C to optical density (OD)600 = 0.5. Cells were

incubated on ice for 15 min prior to centrifugation at 2400 ×g for 5 min at 4°C (Beckman

Avanti® J-25 High Speed Centrifuge, Beckman Coulter, Brea, CA, USA). Cell pellets

were gently resuspended and incubated for 15 min on ice in 0.4× original volume of TfbI solution. After repeated centrifugation, cells were resuspended in 0.04× original volume of TfbII solution and incubated 15 min on ice prior to aliquoting and immediate -80°C storage. Buffer and reagent composition are listed in Table 12.

2.10.3.2 Chemical competent E. coli preparation (calcium chloride; CaCl2)

E. coli strains were inoculated 1:100 from stationary phase (O/N growth at 37°C) into LB

broth and grown aerobically at 37°C to OD600 = 0.5. Cells were incubated on ice for 10

min prior to centrifugation at 4000 × g for 5 min at 4°C (Beckman Avanti® J-25 High

Speed Centrifuge, Beckman Coulter, Brea, CA, USA). Cell pellets were gently

resuspended in 10 mL of cold 0.1 M CaCl2 and incubated for 20 min on ice. After

repeated centrifugation, cells were gently resuspended in 5 mL cold 0.1 M CaCl2 with

2.10.3.3 Electrocompetent E. coli preparation

Electrocompetent cells were prepared with E. coli from stationary phase (O/N growth at 37°C) by subculturing 1:500 into LB media (5 mL) with appropriate antibiotics and grown for 3 h at 37°C, with shaking. The cells were pelleted by centrifugation at 2100 ×

g for 5 min at 4°C and pellet resuspended in 5 mL ice cold 10% glycerol. Centrifugation was repeated and the pellet was resuspended in 1 mL ice cold 10% glycerol followed by a final centrifugation and resuspension in 0.2 mL ice cold 10% glycerol. Cells were aliquoted into 0.1 mL fractions and immediately used for transformation or placed at -80°C for storage.

2.10.4 Transformation and transfection

2.10.4.1 Chemical competent E. coli transformation

To transform chemical competent E. coli, the ligation reaction (~10 µL) was added to the cell aliquot once thawed on ice, and incubated for 30 min remaining on ice. The cells were incubated at 42°C for 45 sec (RbCl2-competent cells) or 2 min (CaCl2-competent

cells) for heat shock and subsequently placed on ice for 2 min. LB culture media (0.9 mL) was added to the cells prior to incubation for 1 h at 37°C with shaking. The transformation was cultured by spread plating (200 µL per plate) on LB Agar with appropriate antibiotics and grown O/N (18-24 h) at 37°C.

2.10.4.2 Electroporation of E. coli

Plasmid DNA (1 µL) was added to electrocompetent E. coli,prepared as described, and incubated on ice for 3 min. Samples were loaded into 0.2 cm electroporation cuvettes (VWR, Radnor, PA, USA) and pulsed at 200 Ω, 25 µF, 2.5 kV for 5 ms (Gene Pulser Xcell™ System, Bio-Rad Laboratories Inc, Hercules, CA, USA). The cells were immediately resuspended in 1 mL of LB culture media and incubated for 1 h at 37°C with shaking. The transformation was cultured by spread plating dilution series from 10-1-10-3 on LB Agar with appropriate antibiotics and grown O/N (18-24 h) at 37°C.

2.10.4.3 Cellular transfections for gene expression

HEK293 cells were seeded as required into multi-well tissue culture plates (Corning Inc, Corning, NY, USA) with cMEM + 10% FBS and allowed to grow O/N (18-24 h) at 37°C with 5% CO2. Liposome:DNA complexes were formed using Lipofectamine® 2000 (Life

Technologies Inc, Carlsbad, CA, USA) and plasmid DNA of choice, as per manufacturer’s protocol at a ratio of 2 µl lipofectamine:0.8 µg DNA. In brief, complexes were formed in cMEM without FBS or antibiotics. Transfection of cells occurred in the same media for 4 h at 37°C with 5% CO2, after which the media was removed and

replaced with cMEM + 10% FBS for plasmid expression over 24 h.

2.10.5 E. coli clone selection

Clones were screened for successful DNA ligation and transformation by selecting individual colonies that were subsequently grown O/N and plasmid isolation performed. Plasmids were visualized for band shifts on 1% agarose gels as described, as well as the insert size verified by restriction enzyme digestion and DNA sequencing performed for final sequence confirmation. Alternatively, DNA screening was performed by colony PCR. Individual colonies were inoculated into single PCR tubes by a sterile toothpick and the reaction buffer (as described) containing T7 forward and T7 terminator primers added. Amplification was performed with the following standard conditions: initial denaturing at 95°C for 10 min followed by repeated cycles (36×) of denaturing at 95°C for 30 sec, annealing with primer-specific temperature 63°C for 30 sec, extension at 74°C at 1 kb min-1, and maintained at 4°C until further processing. PCR products were visualized by electrophoresis and staining with EtBr as described.