47
3 Methods
1. Prepare 100 μg of plasmid DNA containing the gene encoding
the acceptor domain (see Note 1).
2. Prepare a 50 μL working solution of DNase I (1 U/mL) in a
0.5 mL microcentrifuge tube by combining 50 U DNase I,
25 mM Tris–HCl pH 7.5 and 50% glycerol. Store at −20 °C
(see Note 2).
3. Prepare 1.2 mL of diluent solution in a 1.5 mL microcentrifuge tube with a final concentration of 50 mM Tris–HCl pH
7.5, 1 mM MnCl 2 and 0.5× of BSA. Vortex to mix and store at
room temperature (see Note 3).
4. Before creating a library, determine the concentration of DNase
I that will produce the most DNA vectors with a single doublestranded break. To determine this optimum concentration,
combine 20 μg of plasmid DNA and 380 μL of the diluent solution in a 1.5 mL microcentrifuge tube. Mix and centrifuge
briefly to collect all the liquid at the bottom of the tube. Aliquot
95 μL each into four 0.5 mL microcentrifuge tubes and incubate these tubes at 22 °C for 10 min.
5. After the incubation, add 5 μL of diluted DNase I to each tube
at different concentrations (i.e., 1, 2, 4, and 8 mU) and incubate at 22 °C for 8 min.
6. To inactivate the DNase I, add 2.4 μL of 0.5 M EDTA to each
tube and incubate at 75 °C for 10 min.
7. Purify each reaction using the Zymo DNA Clean and
Concentrator kit or a PCR purification kit column. Elute with
water.
8. Run 5–10 μL of each sample on a 0.8% agarose gel made with
1× TAE buffer to determine the concentration of DNase I that
yields the best digestion results (see Notes 4 and 5).
9. Once the optimal DNase I dilution is determined, prepare
80 μg plasmid DNA in 1520 mL of diluent solution and aliquot
95 μL into 16 microtubes (i.e., 5 μg DNA per tube). Incubate
for 10 min at 22 °C.
10. Add 5 μL of the correct DNase dilution to each tube in 30 s
intervals (it will take 8 min to add DNase I to all tubes). After
8 min, stop the reaction as described in step 6 (see Note 6).
11. Combine all the reactions into a 15 mL Nalgene test tube and
purify the DNA using four 25-μg columns from a Zymo DNA
Clean and Concentrator Kit. Elute the DNA from each column
in two 20 μL volumes of water pre-warmed to 65 °C. Combine
the eluate from each spin column (160 μL total) (see Note 7).
3.1 Preparing
Acceptor Vector Using
DNase I
Engineering Protein Switches by Domain Insertion
3 Methods
1. Prepare 100 μg of plasmid DNA containing the gene encoding
the acceptor domain (see Note 1).
2. Prepare a 50 μL working solution of DNase I (1 U/mL) in a
0.5 mL microcentrifuge tube by combining 50 U DNase I,
25 mM Tris–HCl pH 7.5 and 50% glycerol. Store at −20 °C
(see Note 2).
3. Prepare 1.2 mL of diluent solution in a 1.5 mL microcentrifuge tube with a final concentration of 50 mM Tris–HCl pH
7.5, 1 mM MnCl 2 and 0.5× of BSA. Vortex to mix and store at
room temperature (see Note 3).
4. Before creating a library, determine the concentration of DNase
I that will produce the most DNA vectors with a single doublestranded break. To determine this optimum concentration,
combine 20 μg of plasmid DNA and 380 μL of the diluent solution in a 1.5 mL microcentrifuge tube. Mix and centrifuge
briefly to collect all the liquid at the bottom of the tube. Aliquot
95 μL each into four 0.5 mL microcentrifuge tubes and incubate these tubes at 22 °C for 10 min.
5. After the incubation, add 5 μL of diluted DNase I to each tube
at different concentrations (i.e., 1, 2, 4, and 8 mU) and incubate at 22 °C for 8 min.
6. To inactivate the DNase I, add 2.4 μL of 0.5 M EDTA to each
tube and incubate at 75 °C for 10 min.
7. Purify each reaction using the Zymo DNA Clean and
Concentrator kit or a PCR purification kit column. Elute with
water.
8. Run 5–10 μL of each sample on a 0.8% agarose gel made with
1× TAE buffer to determine the concentration of DNase I that
yields the best digestion results (see Notes 4 and 5).
9. Once the optimal DNase I dilution is determined, prepare
80 μg plasmid DNA in 1520 mL of diluent solution and aliquot
95 μL into 16 microtubes (i.e., 5 μg DNA per tube). Incubate
for 10 min at 22 °C.
10. Add 5 μL of the correct DNase dilution to each tube in 30 s
intervals (it will take 8 min to add DNase I to all tubes). After
8 min, stop the reaction as described in step 6 (see Note 6).
11. Combine all the reactions into a 15 mL Nalgene test tube and
purify the DNA using four 25-μg columns from a Zymo DNA
Clean and Concentrator Kit. Elute the DNA from each column
in two 20 μL volumes of water pre-warmed to 65 °C. Combine
the eluate from each spin column (160 μL total) (see Note 7).
3.1 Preparing
Acceptor Vector Using
DNase I
Engineering Protein Switches by Domain Insertion