If an affinity or detection tag needs to be fused to one of the
target genes, the tag coding sequence can be added to the gene
with nested PCRs, or a modified transfer plasmid whose multiple
cloning site already contains the tag sequence can be used. The
plasmid and primer sequences at junctions of a dual expression
cassette for co-expression of eGFP and mCherry fused to a
C-terminal histidine tag are shown Fig. 5.
3.3 Preparation
of the Linearized
Vector and of the DNA
Fragments
The linearized vector and the DNA fragments to be assembled are
prepared by PCR, except when the expression cassette is to be
cloned into large transfer vectors such as pAC8_pMF derivatives.
1. Set up 50 μL PCR to amplify the promoter and backbone
regions of the plasmid and the two genes (Table 3). Use
1–50 ng of template DNA if you amplify your fragments
from a plasmid. Use 100–200 ng of template if you amplify
your genes from cDNA.
2. Run the PCR in a thermocycler by using the conditions suggested by the polymerase manufacturer. We typically denature
the template DNA at 98
C for 30 s, perform 25 cycles of
amplification (denaturation at 98
C for 10 s, annealing at the
calculated Tm for 15 s, and elongation at 72
C for 30 s in the
case of a 1 kb gene) followed by a final incubation at 72
C for
5 min.
3. Load 1/10 of the PCR volume on a 1% agarose gel in parallel
with a molecular weight marker to verify that the PCR amplification resulted in a single band of the expected size.
4. If the fragments are amplified from a plasmid, add ten units of
the DpnI restriction enzyme to the PCR mix and incubate at
37
C for 1 h. DpnI will digest the (methylated) plasmid
template.
5. Purify the PCR on PCR clean-up columns following the
instruction manual, elute bound DNA in 30 μL, and
determine the concentration of the extracted DNA
spectrophotometrically.
We usually prepare between 1 and 2 μg of each fragment. Do
not hesitate to optimize PCR conditions (polymerase buffer,
annealing temperature, addition of DMSO, ) or redesign PCR
primers if yield or PCR specificity are not sufficient (see Note 3).
If DNA fragments are to be cloned into large transfer vectors
such as pAC8_pMF derivatives, the backbone DNA is prepared by
XbaI/NheI restriction and gel purification:
1. Digest 3–5 μg of plasmid in a total volume of 30 μL for 3 h at
37
C with ten units of NheI-HF and XbaI in CutSmart Buffer
(New England Biolabs).
28
Paola Rossolillo et al.
target genes, the tag coding sequence can be added to the gene
with nested PCRs, or a modified transfer plasmid whose multiple
cloning site already contains the tag sequence can be used. The
plasmid and primer sequences at junctions of a dual expression
cassette for co-expression of eGFP and mCherry fused to a
C-terminal histidine tag are shown Fig. 5.
3.3 Preparation
of the Linearized
Vector and of the DNA
Fragments
The linearized vector and the DNA fragments to be assembled are
prepared by PCR, except when the expression cassette is to be
cloned into large transfer vectors such as pAC8_pMF derivatives.
1. Set up 50 μL PCR to amplify the promoter and backbone
regions of the plasmid and the two genes (Table 3). Use
1–50 ng of template DNA if you amplify your fragments
from a plasmid. Use 100–200 ng of template if you amplify
your genes from cDNA.
2. Run the PCR in a thermocycler by using the conditions suggested by the polymerase manufacturer. We typically denature
the template DNA at 98
C for 30 s, perform 25 cycles of
amplification (denaturation at 98
C for 10 s, annealing at the
calculated Tm for 15 s, and elongation at 72
C for 30 s in the
case of a 1 kb gene) followed by a final incubation at 72
C for
5 min.
3. Load 1/10 of the PCR volume on a 1% agarose gel in parallel
with a molecular weight marker to verify that the PCR amplification resulted in a single band of the expected size.
4. If the fragments are amplified from a plasmid, add ten units of
the DpnI restriction enzyme to the PCR mix and incubate at
37
C for 1 h. DpnI will digest the (methylated) plasmid
template.
5. Purify the PCR on PCR clean-up columns following the
instruction manual, elute bound DNA in 30 μL, and
determine the concentration of the extracted DNA
spectrophotometrically.
We usually prepare between 1 and 2 μg of each fragment. Do
not hesitate to optimize PCR conditions (polymerase buffer,
annealing temperature, addition of DMSO, ) or redesign PCR
primers if yield or PCR specificity are not sufficient (see Note 3).
If DNA fragments are to be cloned into large transfer vectors
such as pAC8_pMF derivatives, the backbone DNA is prepared by
XbaI/NheI restriction and gel purification:
1. Digest 3–5 μg of plasmid in a total volume of 30 μL for 3 h at
37
C with ten units of NheI-HF and XbaI in CutSmart Buffer
(New England Biolabs).
28
Paola Rossolillo et al.