48
12. Determine the DNA concentration using a NanoDrop and
run 100 ng of DNA in an agarose gel to estimate the percent
of linear DNA using a gel-imaging software (see Note 4).
13. Once the concentration is known, proceed to the repair step
(see Note 8). Prepare multiple reactions consisting of DNA,
T4 DNA ligase (160 cohesive end units/μg linear DNA), T4
DNA Polymerase (1 unit/μg linear DNA), 200 μM dNTPs,
1× T4 DNA Ligase buffer, and 1× BSA in water. Each reaction
should be <50 μL. Incubate at 12 °C for 20 min.
14. Stop the reaction with 10 mM EDTA (final concentration)
followed by incubation at 75 °C for 10 min.
15. Purify the DNA using a 25-μg Zymo DNA Clean and
Concentrator Kit.
16. Separate the linear DNA from the supercoiled and nicked
DNA via DNA gel electrophoresis. Run the gel at 90 V on a
0.8% agarose gel made with 1× TAE buffer for approximately
1.5 h or until the DNA is separated into three distinct bands
(see Note 9).
17. Excise the band containing the linear DNA and purify using
Invitrogen’s Pure Link Gel Extraction Kit according the manufacturer’s instructions.
18. Dephosphorylate the repaired linear plasmid using Antarctic
Phosphatase. For every microgram of DNA, add 20 units of
enzyme. Incubate for 1 h at 37 °C.
19. Heat inactivate the enzyme by incubating the reaction at 65 °C
for 10 min.
1. Prepare 50 μg of plasmid DNA containing the gene encoding
the acceptor domain (see Note 1).
2. In a 1.5 mL microcentrifuge tube, add 26 μg of plasmid DNA,
1× S1 Nuclease buffer, and 125 units of S1 Nuclease to a final
volume of 325 μL. Aliquot 25 μL each into 13 microtubes
(i.e., 2 μg DNA per tube). Incubate at 37 °C for 20 min.
3. Combine all the reactions and purify using two 25-μg spin columns of a Zymo DNA Clean and Concentrator Kit. Elute the
DNA from each column into two 20 μL aliquots of water prewarmed to 65 °C. Combine the eluate from each spin column
(80 μL total) (see Note 7).
4. Determine the DNA concentration using a NanoDrop and electrophorese 100 ng of sample to estimate the percentage of linear
DNA using gel-imaging software (see Note 4).
5. Once the concentration is known, proceed to the repair, gel
purification, and dephosphorylation steps (see Subheading 3.1,
steps 13–19).
3.2 Preparing
Acceptor Vector Using
S1 Nuclease
Lucas F. Ribeiro et al.
12. Determine the DNA concentration using a NanoDrop and
run 100 ng of DNA in an agarose gel to estimate the percent
of linear DNA using a gel-imaging software (see Note 4).
13. Once the concentration is known, proceed to the repair step
(see Note 8). Prepare multiple reactions consisting of DNA,
T4 DNA ligase (160 cohesive end units/μg linear DNA), T4
DNA Polymerase (1 unit/μg linear DNA), 200 μM dNTPs,
1× T4 DNA Ligase buffer, and 1× BSA in water. Each reaction
should be <50 μL. Incubate at 12 °C for 20 min.
14. Stop the reaction with 10 mM EDTA (final concentration)
followed by incubation at 75 °C for 10 min.
15. Purify the DNA using a 25-μg Zymo DNA Clean and
Concentrator Kit.
16. Separate the linear DNA from the supercoiled and nicked
DNA via DNA gel electrophoresis. Run the gel at 90 V on a
0.8% agarose gel made with 1× TAE buffer for approximately
1.5 h or until the DNA is separated into three distinct bands
(see Note 9).
17. Excise the band containing the linear DNA and purify using
Invitrogen’s Pure Link Gel Extraction Kit according the manufacturer’s instructions.
18. Dephosphorylate the repaired linear plasmid using Antarctic
Phosphatase. For every microgram of DNA, add 20 units of
enzyme. Incubate for 1 h at 37 °C.
19. Heat inactivate the enzyme by incubating the reaction at 65 °C
for 10 min.
1. Prepare 50 μg of plasmid DNA containing the gene encoding
the acceptor domain (see Note 1).
2. In a 1.5 mL microcentrifuge tube, add 26 μg of plasmid DNA,
1× S1 Nuclease buffer, and 125 units of S1 Nuclease to a final
volume of 325 μL. Aliquot 25 μL each into 13 microtubes
(i.e., 2 μg DNA per tube). Incubate at 37 °C for 20 min.
3. Combine all the reactions and purify using two 25-μg spin columns of a Zymo DNA Clean and Concentrator Kit. Elute the
DNA from each column into two 20 μL aliquots of water prewarmed to 65 °C. Combine the eluate from each spin column
(80 μL total) (see Note 7).
4. Determine the DNA concentration using a NanoDrop and electrophorese 100 ng of sample to estimate the percentage of linear
DNA using gel-imaging software (see Note 4).
5. Once the concentration is known, proceed to the repair, gel
purification, and dephosphorylation steps (see Subheading 3.1,
steps 13–19).
3.2 Preparing
Acceptor Vector Using
S1 Nuclease
Lucas F. Ribeiro et al.