50 μL reactions for 2 h at 37
C according to the manufacturer’s instructions.
2. Add 1 μL calf intestinal phosphatase to the pTRA.PVX.
mCherry-2A-CP NheI digest and incubate for 1 h at 37
C.
3. Purify the digested pTRA.PVX.DsRed-2A-CP with a PCR
cleanup kit according to the manufacturer’s instructions.
4. Prepare a 2% agarose gel containing 0.05 μL/mL ethidium
bromide.
5. Apply the entire pMA-RQ.NheI-2xcsy-NheI NheI digest to the
gel, perform electrophoresis, and excise the 56 bp NheI-2xcsyNheI tag band.
6. Purify the NheI-2xcsy-NheI tag DNA from the excised gel slice
using a gel extraction kit, eluting in the smallest volume possible, according to the manufacturer’s instructions.
7. Ligate the NheI-2xcsy-NheI tag into the linearized, dephosphorylated pTRA.PVX.mCherry-2A-CP, using a 10:1 molar
insert-to-vector ratio (see Note 6) in a total volume of 20 μL
with T4 DNA ligase according to the manufacturer’s instructions (see Note 7).
8. Transform 50 μL XL1 Blue MRF’ cells with 3 μL of the ligation
reaction by electroporation, and then incubate cells in 1 mL LB
medium without antibiotic at 37
C for 1 h.
9. Plate 100 μL of the transformation on LB-agar plates containing 100 μg/mL ampicillin, and incubate overnight at 37
C.
10. The next day, inoculate several 5 mL aliquots of LB liquid
medium containing 100 μg/mL ampicillin with a wellseparated colony each, and incubate overnight at 37
C with
shaking.
11. Purify pTRA.PVX.[2xcsy] n .mCherry-2A-CP (see Fig. 1b) plasmids from each culture using a plasmid miniprep kit according
to the manufacturer’s instructions.
12. Set up EcoRI restriction digests with 2.5 μL of each plasmid
DNA in 20 μL total volume according to the manufacturer’s
instructions and incubate at 37
C for 1 h.
13. Prepare a 1% agarose gel containing 0.05 μL/mL ethidium
bromide.
14. Separate the whole DNA restriction reaction by agarose gel
electrophoresis. Plasmid pTRA.PVX.mCherry-2A-CP should
fragment into approximately 0.8, 2.6, and 10.7 kb bands. The
insertion of one or more 2xcsy tags will cause the further
fragmentation of the 10.7 kb band into approximately 2.1
and 7.7 kb products, which are diagnostic for successful
insertions.
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David Burnett et al.
C according to the manufacturer’s instructions.
2. Add 1 μL calf intestinal phosphatase to the pTRA.PVX.
mCherry-2A-CP NheI digest and incubate for 1 h at 37
C.
3. Purify the digested pTRA.PVX.DsRed-2A-CP with a PCR
cleanup kit according to the manufacturer’s instructions.
4. Prepare a 2% agarose gel containing 0.05 μL/mL ethidium
bromide.
5. Apply the entire pMA-RQ.NheI-2xcsy-NheI NheI digest to the
gel, perform electrophoresis, and excise the 56 bp NheI-2xcsyNheI tag band.
6. Purify the NheI-2xcsy-NheI tag DNA from the excised gel slice
using a gel extraction kit, eluting in the smallest volume possible, according to the manufacturer’s instructions.
7. Ligate the NheI-2xcsy-NheI tag into the linearized, dephosphorylated pTRA.PVX.mCherry-2A-CP, using a 10:1 molar
insert-to-vector ratio (see Note 6) in a total volume of 20 μL
with T4 DNA ligase according to the manufacturer’s instructions (see Note 7).
8. Transform 50 μL XL1 Blue MRF’ cells with 3 μL of the ligation
reaction by electroporation, and then incubate cells in 1 mL LB
medium without antibiotic at 37
C for 1 h.
9. Plate 100 μL of the transformation on LB-agar plates containing 100 μg/mL ampicillin, and incubate overnight at 37
C.
10. The next day, inoculate several 5 mL aliquots of LB liquid
medium containing 100 μg/mL ampicillin with a wellseparated colony each, and incubate overnight at 37
C with
shaking.
11. Purify pTRA.PVX.[2xcsy] n .mCherry-2A-CP (see Fig. 1b) plasmids from each culture using a plasmid miniprep kit according
to the manufacturer’s instructions.
12. Set up EcoRI restriction digests with 2.5 μL of each plasmid
DNA in 20 μL total volume according to the manufacturer’s
instructions and incubate at 37
C for 1 h.
13. Prepare a 1% agarose gel containing 0.05 μL/mL ethidium
bromide.
14. Separate the whole DNA restriction reaction by agarose gel
electrophoresis. Plasmid pTRA.PVX.mCherry-2A-CP should
fragment into approximately 0.8, 2.6, and 10.7 kb bands. The
insertion of one or more 2xcsy tags will cause the further
fragmentation of the 10.7 kb band into approximately 2.1
and 7.7 kb products, which are diagnostic for successful
insertions.
168
David Burnett et al.