3.1.2 Insertion
of Mutagenized AGO into
Binary Expression Vector
1. Digest 2 μg of the mutagenized pGEM-AGO plasmid with
appropriate restriction enzymes (see Note 8).
2. Resolve the digested plasmid on a 1.2% agarose gel and purify
the AGO ORF-containing restriction fragment using a DNA
fragment purification kit.
3. Mix the isolated AGO ORF-containing restriction fragment
with pBIN61 plasmid linearized with the appropriate restriction enzymes in a 3:1 molar ratio. Set up a 10 μL ligation
reaction by adding 1 μL of 10Â T4 DNA ligase buffer, appropriate amount of water, and 1 μL (5 U) of T4 DNA ligase to the
above mixture. Incubate the reaction at room temperature for
2–4 h.
4. Competent TOP10 E. coli cells are transformed with the ligation mixture. Spread bacteria onto LB agar plates containing
50 μg/mL kanamycin and incubate plates overnight at 37
C.
5. Screen the appearing colonies by colony PCR (see Note 9).
6. Purify plasmids from colonies which carry the AGO ORF.
Verify the structure of plasmids by restriction digestion.
3.2 Generation
of miRNA Expression
Constructs
The draft genome sequence of N. benthamiana is available from
several sources [11–13]. Based on these data, forward and reverse
primers can be designed to isolate conserved miRNA genes of
N. benthamiana. In order to ensure efficient processing of the
cloned miRNA, in addition to the region encoding the miRNA
hairpin, sufficiently long flanking regions should also be included in
the amplified DNA fragment (at least 200–300 nt in both 5
0 and 3
0
directions).
3.2.1 Isolation
and Cloning of miRNA
Genes of N. benthamiana
1. Isolate plant genomic DNA from young N. benthamiana
leaves using DNeasy Plant Mini Kit.
2. Quantify genomic DNA by nanodrop spectrophotometry.
3. Set up a 50 μL PCR mixture by mixing 100 ng of genomic
DNA, 10 μL of 5Â Phusion buffer, 1 μL of 10 μM forward
primer, 1 μL of 10 μM reverse primer, 1 μL of 10 mM dNTP
mix, appropriate amount of water, and finally 0.5 μL of Phusion polymerase. Place tube into a thermocycler and apply the
following temperature cycling conditions: 98
C for 2 min
(initial denaturation); 35 cycles of 98
C for 10 s (denaturation), Tm + 3
C of the lower Tm primer for 20 s (annealing),
and 72
C for 30 s/kb of fragment to be amplified (extension);
and 72
C for 5 min (polishing).
4. Test an aliquot (1/10th) of the PCR mixture on a 1.2% agarose
TBE gel. Use the rest of the sample to purify the amplified
product by a PCR purification kit.
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of Mutagenized AGO into
Binary Expression Vector
1. Digest 2 μg of the mutagenized pGEM-AGO plasmid with
appropriate restriction enzymes (see Note 8).
2. Resolve the digested plasmid on a 1.2% agarose gel and purify
the AGO ORF-containing restriction fragment using a DNA
fragment purification kit.
3. Mix the isolated AGO ORF-containing restriction fragment
with pBIN61 plasmid linearized with the appropriate restriction enzymes in a 3:1 molar ratio. Set up a 10 μL ligation
reaction by adding 1 μL of 10Â T4 DNA ligase buffer, appropriate amount of water, and 1 μL (5 U) of T4 DNA ligase to the
above mixture. Incubate the reaction at room temperature for
2–4 h.
4. Competent TOP10 E. coli cells are transformed with the ligation mixture. Spread bacteria onto LB agar plates containing
50 μg/mL kanamycin and incubate plates overnight at 37
C.
5. Screen the appearing colonies by colony PCR (see Note 9).
6. Purify plasmids from colonies which carry the AGO ORF.
Verify the structure of plasmids by restriction digestion.
3.2 Generation
of miRNA Expression
Constructs
The draft genome sequence of N. benthamiana is available from
several sources [11–13]. Based on these data, forward and reverse
primers can be designed to isolate conserved miRNA genes of
N. benthamiana. In order to ensure efficient processing of the
cloned miRNA, in addition to the region encoding the miRNA
hairpin, sufficiently long flanking regions should also be included in
the amplified DNA fragment (at least 200–300 nt in both 5
0 and 3
0
directions).
3.2.1 Isolation
and Cloning of miRNA
Genes of N. benthamiana
1. Isolate plant genomic DNA from young N. benthamiana
leaves using DNeasy Plant Mini Kit.
2. Quantify genomic DNA by nanodrop spectrophotometry.
3. Set up a 50 μL PCR mixture by mixing 100 ng of genomic
DNA, 10 μL of 5Â Phusion buffer, 1 μL of 10 μM forward
primer, 1 μL of 10 μM reverse primer, 1 μL of 10 mM dNTP
mix, appropriate amount of water, and finally 0.5 μL of Phusion polymerase. Place tube into a thermocycler and apply the
following temperature cycling conditions: 98
C for 2 min
(initial denaturation); 35 cycles of 98
C for 10 s (denaturation), Tm + 3
C of the lower Tm primer for 20 s (annealing),
and 72
C for 30 s/kb of fragment to be amplified (extension);
and 72
C for 5 min (polishing).
4. Test an aliquot (1/10th) of the PCR mixture on a 1.2% agarose
TBE gel. Use the rest of the sample to purify the amplified
product by a PCR purification kit.
438
Ka ´ roly Fa ´ tyol