49
1. Design one set of primers for each insertion site (see Fig. 2c
and Note 10).
2. Purify the plasmid using the Qiagen Plasmid Miniprep Kit
following the manufacturer’s instructions.
3. Measure the concentration of the DNA using a NanoDrop
(see Note 11).
4. Dilute the template DNA to 10 ng/μL in DNase-free water.
You will need 1 μL per PCR reaction.
5. Prepare 10 μM primer mixes in a 96-well plate (one primer mix
for each primer pair). To each well, add 10 μL of 100 μM forward primer, 10 μL of 100 μM reverse primer, and 80 μL of
water to bring the primer mix to 100 μL total (see Note 12).
6. Prepare a dilute DNA solution by mixing the template DNA
and DNase-free water in a 1.5 mL microcentrifuge tube. For
every PCR reaction, add 8 μL of water and 1 μL of the diluted
DNA stock solution. A separate PCR reaction is required for
each primer mix.
7. To each well of a 96-well PCR plate, add 9 μL of the diluted
DNA solution, 1 μL of primer mix, and 10 μL of Phusion 2×
Master Mix. Decrease the volume setting on the pipette and
mix by pipetting up and down several times (see Note 13).
8. Centrifuge for 30 s at 720 × g to collect all reaction components at the bottom of the wells.
9. Use the following guidelines to design your amplification program (see Note 14):
●
●
1 cycle: 98 °C for 2.5 min.
●
●
25 cycles: (1) 98 °C for 30 s, (2) T opt for 30 s, and (3)
72 °C for 30 s/kilobases of your vector.
●
●
1 cycle: 72 °C for 10 min.
10. Analyze 5 μL from 20 or more random samples using DNA gel
electrophoresis with a 0.8% agarose gel at 90 V for 40 min to
confirm successful amplification of most reactions.
11. After confirmation, pool together 5 μL from each PCR reaction. Using DNA electrophoresis and a 0.8% agarose gel, electrophorese your sample at 90 V for 1 h to allow separation of
the template DNA and PCR product.
12. Excise the PCR product from the gel and purify using
Invitrogen’s PureLink Gel Extraction Kit.
13. Check the concentration and quality of the DNA using a
Nanodrop. If the 260/280 and 260/230 values are too low,
purify the DNA sample using the Zymo Clean and Concentrate
Kit and elute in 20 μL of DNase-free water (see Note 11).
3.3 Preparing
Acceptor Vector Using
Multiplex Inverse PCR
Engineering Protein Switches by Domain Insertion
1. Design one set of primers for each insertion site (see Fig. 2c
and Note 10).
2. Purify the plasmid using the Qiagen Plasmid Miniprep Kit
following the manufacturer’s instructions.
3. Measure the concentration of the DNA using a NanoDrop
(see Note 11).
4. Dilute the template DNA to 10 ng/μL in DNase-free water.
You will need 1 μL per PCR reaction.
5. Prepare 10 μM primer mixes in a 96-well plate (one primer mix
for each primer pair). To each well, add 10 μL of 100 μM forward primer, 10 μL of 100 μM reverse primer, and 80 μL of
water to bring the primer mix to 100 μL total (see Note 12).
6. Prepare a dilute DNA solution by mixing the template DNA
and DNase-free water in a 1.5 mL microcentrifuge tube. For
every PCR reaction, add 8 μL of water and 1 μL of the diluted
DNA stock solution. A separate PCR reaction is required for
each primer mix.
7. To each well of a 96-well PCR plate, add 9 μL of the diluted
DNA solution, 1 μL of primer mix, and 10 μL of Phusion 2×
Master Mix. Decrease the volume setting on the pipette and
mix by pipetting up and down several times (see Note 13).
8. Centrifuge for 30 s at 720 × g to collect all reaction components at the bottom of the wells.
9. Use the following guidelines to design your amplification program (see Note 14):
●
●
1 cycle: 98 °C for 2.5 min.
●
●
25 cycles: (1) 98 °C for 30 s, (2) T opt for 30 s, and (3)
72 °C for 30 s/kilobases of your vector.
●
●
1 cycle: 72 °C for 10 min.
10. Analyze 5 μL from 20 or more random samples using DNA gel
electrophoresis with a 0.8% agarose gel at 90 V for 40 min to
confirm successful amplification of most reactions.
11. After confirmation, pool together 5 μL from each PCR reaction. Using DNA electrophoresis and a 0.8% agarose gel, electrophorese your sample at 90 V for 1 h to allow separation of
the template DNA and PCR product.
12. Excise the PCR product from the gel and purify using
Invitrogen’s PureLink Gel Extraction Kit.
13. Check the concentration and quality of the DNA using a
Nanodrop. If the 260/280 and 260/230 values are too low,
purify the DNA sample using the Zymo Clean and Concentrate
Kit and elute in 20 μL of DNase-free water (see Note 11).
3.3 Preparing
Acceptor Vector Using
Multiplex Inverse PCR
Engineering Protein Switches by Domain Insertion