52
in length, (3) the last two nucleotides on the 3′ each of primer
should be Cs or Gs, and (4) there should be no more than 3
Cs or Gs in the final five nucleotides of the 3′ end of the primer.
All primers should be designed such that they have approximately the same melting temperature (ideally 60–65 ºC). The 5′
end of the forward primer should begin at the codon downstream of the insertion site. The 5′ end of the reverse primer
should begin at last nucleotide of the codon that is upstream of
the insertion site. For example, if you want to insert a gene
between codons 2 and 3 of the acceptor DNA, the 5′ end of
your forward primer should begin at the first nucleotide in
codon 3 and the 5′ end of your reverse primer should begin at
the last nucleotide in codon 2. This will create an in-frame
opening between codons 2 and 3 (see Fig. 2d). You can also
duplicate and delete codons by shifting the primers upstream
or downstream (see Fig. 2d). We have written a MATLAB
script to design such primer pairs, which is freely available.
After designing the primers, use a PCR simulation software
such as AmplifX to determine the optimal annealing temperature (T opt ) for a few of the primer pairs. Use the lowest T opt
value of the primer pairs. Also use the software to check for the
likelihood of nonspecific primer annealing to the template or
the formation of primer dimers. Alternatively, you can subtract
3–4 °C from the average melting temperature of all the primers
as a good estimation for T opt .
11. Check the quality of your DNA. A reading with 260/280
value of 1.8–2.0 and a 260/230 value of 1.8–2.3 will yield the
best PCR results.
12. You can scale down the volume of the primer mix if necessary.
13. This process can be facilitated by using 12-channel basins and
12-channel pipettes.
14. You can minimize the formation of undesired PCR product by
performing touch-down PCR using the following amplification
program: (i) Preheat the thermocycler to 98 °C, (ii) 1 cycle:
98°C for 3 min, (iii) 15 cycles: (1) 98 °C for 30 s, (2)
T opt + 15 °C for 30 s, and (3) 72 °C for 30 s/kb of GOI. The
annealing temperature should decrease by 1 °C for each cycle,
(iv) 10 cycles: (1) 98 °C for 30 s, (2) T opt for 30 s, and (3)
72 °C for 30 s/kb of GOI, (v) 72 °C for 10 min.
15. Circular permutation aims to change the position of N- and
C-termini of the protein without disturbing its threedimensional structure. In general, the N- and C-terminals of
the original protein should be close enough to allow their union
(<10 Å) [19].
16. When the gene (without a stop codon) is already available in
cloning vector and a convenient restriction site is available at
Lucas F. Ribeiro et al.
in length, (3) the last two nucleotides on the 3′ each of primer
should be Cs or Gs, and (4) there should be no more than 3
Cs or Gs in the final five nucleotides of the 3′ end of the primer.
All primers should be designed such that they have approximately the same melting temperature (ideally 60–65 ºC). The 5′
end of the forward primer should begin at the codon downstream of the insertion site. The 5′ end of the reverse primer
should begin at last nucleotide of the codon that is upstream of
the insertion site. For example, if you want to insert a gene
between codons 2 and 3 of the acceptor DNA, the 5′ end of
your forward primer should begin at the first nucleotide in
codon 3 and the 5′ end of your reverse primer should begin at
the last nucleotide in codon 2. This will create an in-frame
opening between codons 2 and 3 (see Fig. 2d). You can also
duplicate and delete codons by shifting the primers upstream
or downstream (see Fig. 2d). We have written a MATLAB
script to design such primer pairs, which is freely available.
After designing the primers, use a PCR simulation software
such as AmplifX to determine the optimal annealing temperature (T opt ) for a few of the primer pairs. Use the lowest T opt
value of the primer pairs. Also use the software to check for the
likelihood of nonspecific primer annealing to the template or
the formation of primer dimers. Alternatively, you can subtract
3–4 °C from the average melting temperature of all the primers
as a good estimation for T opt .
11. Check the quality of your DNA. A reading with 260/280
value of 1.8–2.0 and a 260/230 value of 1.8–2.3 will yield the
best PCR results.
12. You can scale down the volume of the primer mix if necessary.
13. This process can be facilitated by using 12-channel basins and
12-channel pipettes.
14. You can minimize the formation of undesired PCR product by
performing touch-down PCR using the following amplification
program: (i) Preheat the thermocycler to 98 °C, (ii) 1 cycle:
98°C for 3 min, (iii) 15 cycles: (1) 98 °C for 30 s, (2)
T opt + 15 °C for 30 s, and (3) 72 °C for 30 s/kb of GOI. The
annealing temperature should decrease by 1 °C for each cycle,
(iv) 10 cycles: (1) 98 °C for 30 s, (2) T opt for 30 s, and (3)
72 °C for 30 s/kb of GOI, (v) 72 °C for 10 min.
15. Circular permutation aims to change the position of N- and
C-termini of the protein without disturbing its threedimensional structure. In general, the N- and C-terminals of
the original protein should be close enough to allow their union
(<10 Å) [19].
16. When the gene (without a stop codon) is already available in
cloning vector and a convenient restriction site is available at
Lucas F. Ribeiro et al.