50
1. Construct a vector as shown in Fig. 2c by duplicating the insert
gene by end-to-end fusion, connecting them with a linker (see
Notes 15–17).
2. Design a forward primer starting at the first base of the selected
codon and a reverse primer starting at the last base of the previous
codon (the melting temperatures should be between 60 and
65 °C (see Notes 10 and 18–20).
3. The PCR protocol is the same as used for multiplex inverse
PCR (see Subheading 3.3, steps 2–13).
1. Starting with 200 ng of prepared vector, calculate the pmol of
vector using the following formula:
pmol
of
kilobase pairs of vector
= (
)
´
(
)
ng
DNA
D a
/
.
650
2. Using the same formula, calculate the pmol of insert required
to achieve a 3:1 or 5:1 insert-to-vector molar ratio.
3. Add the calculated amount of prepared vector and insert to a
thin-walled PCR tube. Add 2 μL of 10× T4 DNA Ligase Buffer
and fill to 19 μL. Add 1 μL of T4 DNA Ligase, flick the tube
to mix and centrifuge briefly to collect the reaction components at the bottom of the tube (see Notes 21 and 22).
4. Place the ligation reaction in a thermocycler. Incubate for
12–18 h cycling between: 25 °C for 30 s and 10 °C for 30 s.
5. After the incubation, purify the DNA using the Zymo Clean &
Concentrator Kit. Elute the DNA into a 1.5 mL microcentrifuge
tube in 10 μL of water. Concentrate your sample to approximately
4 μL using a vacufuge at room temperature (see Note 23).
1. Turn on the Gene Pulser. Adjust the voltage to 2.5 kV.
2. Place the following on ice: high-efficiency (>10
8
cfu/μg DNA)
electrocompetent cells, a 0.2 cm Gene Pulser Cuvette, a
0.5 mL microcentrifuge tube and your purified and concentrated ligation (see Note 24).
3. Once the cells are thawed, aliquot 40 μL of cells into the
microcentrifuge tube with the ligation, ensuring the cells
engulf the DNA sample. Mix well.
4. Pipette the cells into the cuvette (see Note 25).
5. Place the cuvette in the Gene Pulser and slide into the chamber, ensuring that the cuvette clicks into place.
6. Simultaneously hold down the pulse buttons until you hear the
beep. Then, release the buttons.
7. Quickly, add 1 mL of SOC media to the cuvette.
8. Transfer the cell/SOC mix to a 1.5 mL microcentrifuge tube.
3.4 Preparing
Circular Permuted
Insert
3.5 Ligation
of the Insert
and Acceptor DNA
3.6 Transformation
Lucas F. Ribeiro et al.
1. Construct a vector as shown in Fig. 2c by duplicating the insert
gene by end-to-end fusion, connecting them with a linker (see
Notes 15–17).
2. Design a forward primer starting at the first base of the selected
codon and a reverse primer starting at the last base of the previous
codon (the melting temperatures should be between 60 and
65 °C (see Notes 10 and 18–20).
3. The PCR protocol is the same as used for multiplex inverse
PCR (see Subheading 3.3, steps 2–13).
1. Starting with 200 ng of prepared vector, calculate the pmol of
vector using the following formula:
pmol
of
kilobase pairs of vector
= (
)
´
(
)
ng
DNA
D a
/
.
650
2. Using the same formula, calculate the pmol of insert required
to achieve a 3:1 or 5:1 insert-to-vector molar ratio.
3. Add the calculated amount of prepared vector and insert to a
thin-walled PCR tube. Add 2 μL of 10× T4 DNA Ligase Buffer
and fill to 19 μL. Add 1 μL of T4 DNA Ligase, flick the tube
to mix and centrifuge briefly to collect the reaction components at the bottom of the tube (see Notes 21 and 22).
4. Place the ligation reaction in a thermocycler. Incubate for
12–18 h cycling between: 25 °C for 30 s and 10 °C for 30 s.
5. After the incubation, purify the DNA using the Zymo Clean &
Concentrator Kit. Elute the DNA into a 1.5 mL microcentrifuge
tube in 10 μL of water. Concentrate your sample to approximately
4 μL using a vacufuge at room temperature (see Note 23).
1. Turn on the Gene Pulser. Adjust the voltage to 2.5 kV.
2. Place the following on ice: high-efficiency (>10
8
cfu/μg DNA)
electrocompetent cells, a 0.2 cm Gene Pulser Cuvette, a
0.5 mL microcentrifuge tube and your purified and concentrated ligation (see Note 24).
3. Once the cells are thawed, aliquot 40 μL of cells into the
microcentrifuge tube with the ligation, ensuring the cells
engulf the DNA sample. Mix well.
4. Pipette the cells into the cuvette (see Note 25).
5. Place the cuvette in the Gene Pulser and slide into the chamber, ensuring that the cuvette clicks into place.
6. Simultaneously hold down the pulse buttons until you hear the
beep. Then, release the buttons.
7. Quickly, add 1 mL of SOC media to the cuvette.
8. Transfer the cell/SOC mix to a 1.5 mL microcentrifuge tube.
3.4 Preparing
Circular Permuted
Insert
3.5 Ligation
of the Insert
and Acceptor DNA
3.6 Transformation
Lucas F. Ribeiro et al.