6. Excise the 12xMSL fragment by PacI/SacI digestion and purify
the fragment from an agarose gel.
7. Digest plasmid pMDC32 with PacI/SacI and purify the linearized plasmid from an agarose gel.
8. Ligate the purified 12xMSL fragment into the linearized and
purified pMDC32 plasmid with T4 DNA ligase to create
pMDC32-MS2-12xMSL. Transform E. coli DB3.1 highefficiency competent cells with 1.0–3.0 μL of the ligation mix
and distribute the cells on petri dish with antibiotic selection
(50 μg/mL kanamycin and 34 μg/mL chloramphenicol).
Grow colonies overnight at 37
C.
9. Isolate bacteria from 4 to 5 isolated colonies and propagate
them overnight in 15 mL falcon tubes containing 4.0 mL of
liquid LB medium with antibiotics (50 μg/mL kanamycin and
34 μg/mL chloramphenicol) at 37
C with agitation
(180–200 rpm).
10. Isolate the plasmid DNA (i.e., the destination vector
pMDC32-GW-12xMSL) using a plasmid purification kit and
confirm the correct insertion of the 12xMSL tandem by DNA
sequencing with ccdBfw and T-nosrev primers.
3.1.2 Cloning of the RNA
of Interest into
pMDC32-GW-12xMSL
1. Design primers for PCR amplification of the DNA fragment
encoding your RNA of interest and add attB sequences
required for BP™ cloning (see Note 4).
2. Amplify the DNA fragment encoding your RNA of interest by
PCR using a high-fidelity DNA polymerase and the appropriate
DNA source as template (DNA or cDNA). Verify the correct
size of the amplified DNA fragment by agarose gel electrophoresis and, subsequently, purify the fragment from the gel using
an agarose gel extraction kit.
3. Perform a BP™ recombination reaction between the purified
DNA fragment and pDONR™/Zeo, with the BP™ clonase
enzyme mix (Thermo Fisher) according to the manufacturer’s
instructions, to create your entry clone. Transform E. coli cells
with 0.5–1.0 μL of the reaction and grow colonies overnight at
37
C on LB plates under antibiotic selection (20 μg/mL of
zeocin for pDONR™/Zeo).
4. Use 4–5 colonies for growing bacterial cultures in liquid LB
medium with antibiotic selection (20 μg/mL of zeocin for
pDONR™/Zeo) overnight at 37
C with 180–200 rpm
agitation.
5. Isolate the plasmid DNA (the entry vector), verify the presence,
and correct cloning of your DNA by DNA sequencing (use
universal primers homologous to vector sequences, e.g.,
M13fw and M13rev for pDONR™/Zeo).
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Eduardo Jose ´ Pen ˜ a and Manfred Heinlein
the fragment from an agarose gel.
7. Digest plasmid pMDC32 with PacI/SacI and purify the linearized plasmid from an agarose gel.
8. Ligate the purified 12xMSL fragment into the linearized and
purified pMDC32 plasmid with T4 DNA ligase to create
pMDC32-MS2-12xMSL. Transform E. coli DB3.1 highefficiency competent cells with 1.0–3.0 μL of the ligation mix
and distribute the cells on petri dish with antibiotic selection
(50 μg/mL kanamycin and 34 μg/mL chloramphenicol).
Grow colonies overnight at 37
C.
9. Isolate bacteria from 4 to 5 isolated colonies and propagate
them overnight in 15 mL falcon tubes containing 4.0 mL of
liquid LB medium with antibiotics (50 μg/mL kanamycin and
34 μg/mL chloramphenicol) at 37
C with agitation
(180–200 rpm).
10. Isolate the plasmid DNA (i.e., the destination vector
pMDC32-GW-12xMSL) using a plasmid purification kit and
confirm the correct insertion of the 12xMSL tandem by DNA
sequencing with ccdBfw and T-nosrev primers.
3.1.2 Cloning of the RNA
of Interest into
pMDC32-GW-12xMSL
1. Design primers for PCR amplification of the DNA fragment
encoding your RNA of interest and add attB sequences
required for BP™ cloning (see Note 4).
2. Amplify the DNA fragment encoding your RNA of interest by
PCR using a high-fidelity DNA polymerase and the appropriate
DNA source as template (DNA or cDNA). Verify the correct
size of the amplified DNA fragment by agarose gel electrophoresis and, subsequently, purify the fragment from the gel using
an agarose gel extraction kit.
3. Perform a BP™ recombination reaction between the purified
DNA fragment and pDONR™/Zeo, with the BP™ clonase
enzyme mix (Thermo Fisher) according to the manufacturer’s
instructions, to create your entry clone. Transform E. coli cells
with 0.5–1.0 μL of the reaction and grow colonies overnight at
37
C on LB plates under antibiotic selection (20 μg/mL of
zeocin for pDONR™/Zeo).
4. Use 4–5 colonies for growing bacterial cultures in liquid LB
medium with antibiotic selection (20 μg/mL of zeocin for
pDONR™/Zeo) overnight at 37
C with 180–200 rpm
agitation.
5. Isolate the plasmid DNA (the entry vector), verify the presence,
and correct cloning of your DNA by DNA sequencing (use
universal primers homologous to vector sequences, e.g.,
M13fw and M13rev for pDONR™/Zeo).
112
Eduardo Jose ´ Pen ˜ a and Manfred Heinlein