sequenced. In this case, only the sequenced fragment, bearing
the desired mutation, is swapped into the binary expression
vector using appropriate restriction enzymes.
8. The polylinker region of pGEM-T easy plasmid vector contains
recognition sites for a relatively large number of rare-cutting
restriction enzymes. Various combinations of these enzymes
can be used to isolate the intact AGO ORFs from the vector.
If not sufficient, additional recognition sites can also be added
to the vector using appropriate oligonucleotides.
9. Colony PCR can be used to screen for bacterial colonies carrying plasmids with the desired insert. The protocol is carried out
as follows:
(a) Touch isolated bacterial colonies with a pipet tip and
transfer the attached cells into 20 μL of water (in a
PCR tube).
(b) Lyse bacteria by heating the suspension to 95
C for 5 min
in a thermocycler.
(c) Transfer 0.5 μL of the bacterial lysate into a PCR tube
containing 12 μL of PCR mix. The PCR mix is assembled
as follows: 0.25 μL of 10 μM forward primer, 0.25 μL of
10 μM reverse primer, 0.25 μL of 10 mM dNTP mix,
0.75 μL of 25 mM MgCl 2 , 1.25 μL of 10Â Taq buffer,
9 μL of water, and 0.25 μL of Taq polymerase. By
up-scaling the above volumes, a PCR master mix can be
prepared to screen the desired number of colonies. At least
one of the applied primers should be specific for the
plasmid vector. This helps to eliminate the amplification
of background products, which derive from the unused
insert. Additionally, this allows to determine the orientation of the inserted DNA fragment within the vector.
(d) Use the following temperature cycling program: 94
C for
2 min (initial denaturation); 40 cycles of 94
C for 30 s
(denaturation), Tm
C of the lower Tm primer for 30 s
(annealing), and 72
C for 30 s/kb of the fragment to be
amplified (extension); and 72
C for 7 min (polishing).
(e) Check PCR reactions on a TBE agarose gel (adjust gel
density by changing agarose concentration according to
the size of the expected DNA fragment).
10. Using the specific substrate of Renilla luciferase (coelenterazine), we have demonstrated the functionality of the sensor
plasmid-encoded GFP-Renilla fusion protein. We also found
that the protein’s enzymatic activity correlated perfectly with
the signal intensity measured in quantitative Western blots
using anti-GFP antibody. In addition, we demonstrated that
the protein is suitable for dual-luciferase assays (combined with
firefly luciferase normalization control, unpublished result).
448
Ka ´ roly Fa ´ tyol
the desired mutation, is swapped into the binary expression
vector using appropriate restriction enzymes.
8. The polylinker region of pGEM-T easy plasmid vector contains
recognition sites for a relatively large number of rare-cutting
restriction enzymes. Various combinations of these enzymes
can be used to isolate the intact AGO ORFs from the vector.
If not sufficient, additional recognition sites can also be added
to the vector using appropriate oligonucleotides.
9. Colony PCR can be used to screen for bacterial colonies carrying plasmids with the desired insert. The protocol is carried out
as follows:
(a) Touch isolated bacterial colonies with a pipet tip and
transfer the attached cells into 20 μL of water (in a
PCR tube).
(b) Lyse bacteria by heating the suspension to 95
C for 5 min
in a thermocycler.
(c) Transfer 0.5 μL of the bacterial lysate into a PCR tube
containing 12 μL of PCR mix. The PCR mix is assembled
as follows: 0.25 μL of 10 μM forward primer, 0.25 μL of
10 μM reverse primer, 0.25 μL of 10 mM dNTP mix,
0.75 μL of 25 mM MgCl 2 , 1.25 μL of 10Â Taq buffer,
9 μL of water, and 0.25 μL of Taq polymerase. By
up-scaling the above volumes, a PCR master mix can be
prepared to screen the desired number of colonies. At least
one of the applied primers should be specific for the
plasmid vector. This helps to eliminate the amplification
of background products, which derive from the unused
insert. Additionally, this allows to determine the orientation of the inserted DNA fragment within the vector.
(d) Use the following temperature cycling program: 94
C for
2 min (initial denaturation); 40 cycles of 94
C for 30 s
(denaturation), Tm
C of the lower Tm primer for 30 s
(annealing), and 72
C for 30 s/kb of the fragment to be
amplified (extension); and 72
C for 7 min (polishing).
(e) Check PCR reactions on a TBE agarose gel (adjust gel
density by changing agarose concentration according to
the size of the expected DNA fragment).
10. Using the specific substrate of Renilla luciferase (coelenterazine), we have demonstrated the functionality of the sensor
plasmid-encoded GFP-Renilla fusion protein. We also found
that the protein’s enzymatic activity correlated perfectly with
the signal intensity measured in quantitative Western blots
using anti-GFP antibody. In addition, we demonstrated that
the protein is suitable for dual-luciferase assays (combined with
firefly luciferase normalization control, unpublished result).
448
Ka ´ roly Fa ´ tyol