3. Set up a standard PCR reaction mixture containing 50 ng of
DNA, 0.2 mM of dNTPs, 1 μM of carS-PmeI-1F and 1 μM
carS-PmeI-1R, 1.75 mM of MgCl 2, 1Â PCR buffer, and 1 U of
Expand High Fidelity PCR System.
4. Perform a PCR using a program fitting your primer Tms. For
carS, program was: Denaturalization at 94
C for 2 min.
35 cycles of amplification reactions consisting of 94
C, 20 s;
55
C, 30 s; 72
C, 2 min. Final polymerization step of 10 min
at 72
C.
5. Separate DNA PCR fragment in a 0.8% agarose gel electrophoresis (100 V for 45 min).
6. Purify the carS ORF fragment. Elute DNA in 10 μl of ultrapure
water.
7. Ligate the PmeI hanging carS fragment with vector pGEM-T
easy. Incubate an appropriate DNA amount of insert and 1 μl of
vector pGEM-T easy (50 ng/μl) with 1 U of T4 DNA ligase,
5 μl of 2Â Rapid Ligation Buffer in a final volume of 10 μl.
Incubate overnight at 16
C or 1 h at room temperature.
8. Incubate competent cells of E. coli DH5α with 5 μl of ligation
reaction for 30 min in ice. Transform E. coli by thermic shock
following a standard protocol [22]. Spread transformed bacteria on LB-X-Gal-Amp agar medium and incubate plates overnight at 37
C.
9. Select several white colonies to screen positives clones. Pick
selected colonies to fresh liquid LB-Amp, incubate overnight
at 37
C. Extract plasmid DNA from E. coli clones
following [22].
10. Digest 1 μg of plasmid with 1 U of EcoRI in 1Â H buffer.
Incubate at 37
C during 2 h. Check size of fragments by
electrophoresis in a 0,8% agarose gel in TAE 1Â buffer. The
correct clones gave two bands of 3 kb and 2 kb, corresponding
to the vector pGEM-T and carS gene, respectively. One of the
positives clones was named pPO3.
11. Digest pPO3 and pVG3, a plasmid with the Tet-on based
system [19], with PmeI at 37
C, to release carS ORF from
pPO3 (or gene of interest) and to introduce it in pVG3.
12. Separate DNA fragments by electrophoresis in a 0.8% agarose
gel in TAE 1Â buffer. Include as DNA size marker 5 μl of the
1-kb ladder marker.
13. Cut with a scalpel blade 2 agarose gel slices containing the carS
ORF from pPO5 vector (2 kb) and the large fragment from
plasmid pVG3 (6.7 kb).
14. Purify fragments corresponding to the carS ORF from pPO3
(insert) and to pVG3 without the mluc (vector) with Isolate II
PCR and Gel Kit (Bioline).
Modulated Expression of a Key Regulatory Gene in Carotenoid Biosynthesis
349
DNA, 0.2 mM of dNTPs, 1 μM of carS-PmeI-1F and 1 μM
carS-PmeI-1R, 1.75 mM of MgCl 2, 1Â PCR buffer, and 1 U of
Expand High Fidelity PCR System.
4. Perform a PCR using a program fitting your primer Tms. For
carS, program was: Denaturalization at 94
C for 2 min.
35 cycles of amplification reactions consisting of 94
C, 20 s;
55
C, 30 s; 72
C, 2 min. Final polymerization step of 10 min
at 72
C.
5. Separate DNA PCR fragment in a 0.8% agarose gel electrophoresis (100 V for 45 min).
6. Purify the carS ORF fragment. Elute DNA in 10 μl of ultrapure
water.
7. Ligate the PmeI hanging carS fragment with vector pGEM-T
easy. Incubate an appropriate DNA amount of insert and 1 μl of
vector pGEM-T easy (50 ng/μl) with 1 U of T4 DNA ligase,
5 μl of 2Â Rapid Ligation Buffer in a final volume of 10 μl.
Incubate overnight at 16
C or 1 h at room temperature.
8. Incubate competent cells of E. coli DH5α with 5 μl of ligation
reaction for 30 min in ice. Transform E. coli by thermic shock
following a standard protocol [22]. Spread transformed bacteria on LB-X-Gal-Amp agar medium and incubate plates overnight at 37
C.
9. Select several white colonies to screen positives clones. Pick
selected colonies to fresh liquid LB-Amp, incubate overnight
at 37
C. Extract plasmid DNA from E. coli clones
following [22].
10. Digest 1 μg of plasmid with 1 U of EcoRI in 1Â H buffer.
Incubate at 37
C during 2 h. Check size of fragments by
electrophoresis in a 0,8% agarose gel in TAE 1Â buffer. The
correct clones gave two bands of 3 kb and 2 kb, corresponding
to the vector pGEM-T and carS gene, respectively. One of the
positives clones was named pPO3.
11. Digest pPO3 and pVG3, a plasmid with the Tet-on based
system [19], with PmeI at 37
C, to release carS ORF from
pPO3 (or gene of interest) and to introduce it in pVG3.
12. Separate DNA fragments by electrophoresis in a 0.8% agarose
gel in TAE 1Â buffer. Include as DNA size marker 5 μl of the
1-kb ladder marker.
13. Cut with a scalpel blade 2 agarose gel slices containing the carS
ORF from pPO5 vector (2 kb) and the large fragment from
plasmid pVG3 (6.7 kb).
14. Purify fragments corresponding to the carS ORF from pPO3
(insert) and to pVG3 without the mluc (vector) with Isolate II
PCR and Gel Kit (Bioline).
Modulated Expression of a Key Regulatory Gene in Carotenoid Biosynthesis
349