with 1 μL of each restriction enzyme in a total volume reaction
of 30 μL. DNA digestion with restriction enzymes is typically
performed at 37
C for at least 1 h (see Note 12).
4. Run the plasmid restriction reactions on a 1% agarose-TAE gel
and cut slices of agarose gel around the digested plasmid backbone bands. Extract and purify the digested DNA using a
standard DNA gel extraction kit. Digested PCR products can
be directly purified using a PCR or reaction clean up protocol
usually included in gel extraction kits.
5. Ligate digested plasmids and PCR products using T4 DNA
ligase. Typically, a total of 50–100 ng DNA (plasmid + insert
with a three- to fivefold molar excess insert/plasmid) is mixed
with ligation buffer and DNA ligase in a total of 10 μL and
incubated at room temperature for 5–10 min.
6. Use 3 μL of the ligation reaction to transform 50 μL of competent E. coli cells. Typically bacteria and DNA are gently
mixed and incubated on ice for 30 min. Transformation is
then induced by heat shock at 42
C for 30–45 s. The cells
are then placed back on ice. After 2 min, 250 μL of pre-warmed
(37
C) LB medium is added and the cells incubated on a
shaking block at 37
C for 1 h at 800 rpm (see Note 13).
Thereafter, 100 μL of the incubation is spread on a 10 cm
bacterial LB-agar plate containing the appropriate antibiotic
selection. The plates are incubated overnight at 37
C.
7. On the next day, two to four antibiotic-resistant colonies are
picked to inoculate 3 mL of LB medium containing the appropriate antibiotic in 15 mL tubes. The tubes are then incubated
for 12–16 h at 37
C, 180 rpm in a shaker-incubator.
8. On the next day, plasmids are isolated by Miniprep and their
sequences verified by Sanger sequencing analysis.
9. Correct plasmids can be re-amplified and purified with a Midiprep protocol to generate a larger stock of sequence-verified
split-BioID plasmids.
3.2 Small-Scale
Testing of ProximityDependent
Biotinylation
in Living Cell
1. Seed a six-well cell culture plate with the cells to transfect so
that they reach 50–70% confluence on the following day. Incubate overnight at 37
C, 5% CO 2 . For HeLa cells, typically
80–150 Â 10
3 cells are seeded per well in a volume of 2 mL (see
Note 14).
2. On the next day, remove the medium from the six-well plate
and replace with 2 mL of fresh medium per well.
3. Prepare the transfection mixes for all conditions to be tested,
usually the pair of proteins of interest and a negative control (see
Note 15). When using PEI as a transfection reagent for HeLa
cells, we typically use 6 μg PEI diluted in 100 μL DMEM
308
Cinthia Amaya Ramirez et al.
of 30 μL. DNA digestion with restriction enzymes is typically
performed at 37
C for at least 1 h (see Note 12).
4. Run the plasmid restriction reactions on a 1% agarose-TAE gel
and cut slices of agarose gel around the digested plasmid backbone bands. Extract and purify the digested DNA using a
standard DNA gel extraction kit. Digested PCR products can
be directly purified using a PCR or reaction clean up protocol
usually included in gel extraction kits.
5. Ligate digested plasmids and PCR products using T4 DNA
ligase. Typically, a total of 50–100 ng DNA (plasmid + insert
with a three- to fivefold molar excess insert/plasmid) is mixed
with ligation buffer and DNA ligase in a total of 10 μL and
incubated at room temperature for 5–10 min.
6. Use 3 μL of the ligation reaction to transform 50 μL of competent E. coli cells. Typically bacteria and DNA are gently
mixed and incubated on ice for 30 min. Transformation is
then induced by heat shock at 42
C for 30–45 s. The cells
are then placed back on ice. After 2 min, 250 μL of pre-warmed
(37
C) LB medium is added and the cells incubated on a
shaking block at 37
C for 1 h at 800 rpm (see Note 13).
Thereafter, 100 μL of the incubation is spread on a 10 cm
bacterial LB-agar plate containing the appropriate antibiotic
selection. The plates are incubated overnight at 37
C.
7. On the next day, two to four antibiotic-resistant colonies are
picked to inoculate 3 mL of LB medium containing the appropriate antibiotic in 15 mL tubes. The tubes are then incubated
for 12–16 h at 37
C, 180 rpm in a shaker-incubator.
8. On the next day, plasmids are isolated by Miniprep and their
sequences verified by Sanger sequencing analysis.
9. Correct plasmids can be re-amplified and purified with a Midiprep protocol to generate a larger stock of sequence-verified
split-BioID plasmids.
3.2 Small-Scale
Testing of ProximityDependent
Biotinylation
in Living Cell
1. Seed a six-well cell culture plate with the cells to transfect so
that they reach 50–70% confluence on the following day. Incubate overnight at 37
C, 5% CO 2 . For HeLa cells, typically
80–150 Â 10
3 cells are seeded per well in a volume of 2 mL (see
Note 14).
2. On the next day, remove the medium from the six-well plate
and replace with 2 mL of fresh medium per well.
3. Prepare the transfection mixes for all conditions to be tested,
usually the pair of proteins of interest and a negative control (see
Note 15). When using PEI as a transfection reagent for HeLa
cells, we typically use 6 μg PEI diluted in 100 μL DMEM
308
Cinthia Amaya Ramirez et al.