1. Remove residual ssDNA by ExoI treatment of 0.3 μg of the
PCR amplified oligonucleotide library using 0.2 units/μl,
37
C for 30 min, 85
C for 15 min, followed by flash cooling
on ice.
2. 5’Phosphorylate the DNA directly after flash cooling. Combine in a 1.5 ml micro centrifuge tube the library with 2 μl 10Â
TM buffer, 2 μl 10 mM ATP, and 1 μl 100 mM DTT. Add
water up to 20 μl.
3. Add 10 units of T4 polynucleotide kinase and incubate for 1 h
at 37
C. Use the phosphorylated sample directly for annealing.
4. To 10 μg of dU-ssDNA add 25 μl of 10Â TM buffer, 20 μl of
the phosphorylated oligonucleotides and adjust to a final volume of 250 μl.
5. Incubate at 90
C for 3 min, 50
C for 3 min and 20
C for
5 min.
6. Add 10 μl of 10 mM dNTP, 10 μl 10 mM ATP 15 μl 100 mM
DTT, 30 units T7 DNA polymerase, and 30 Weiss units of T4
DNA ligase to the annealed oligonucleotides.
7. Incubate the reaction for 16 h at 20
C. This will synthesize the
second strand and ligate it to form the heteroduplex
CCC-dsDNA.
8. Freeze-thaw the sample 3 times to inactivate the enzymes.
9. Add 5 μl Fast-digest SmaI to remove phagemid without insert
(see Note 3). Incubate for 30 min at 37
C.
10. Purify and desalt the CCC-dsDNA using QIAquick DNA
purification kit. Add 1 ml of QG buffer to the reaction and mix.
11. Load the sample to two spin columns placed in 2-ml microcentrifuge tubes. Centrifuge at 16,000 Â g for 1 min, discard
the flow through.
12. To each column add 750 μl of PE buffer and spin the sample at
16,000 Â g for 1 min, discard the flow through.
13. Centrifuge the columns an extra time at 16,000 Â g for 1 min
to remove excess PE buffer.
14. Transfer the column to a new 1.5 ml microcentrifuge tube, add
35 μl of ultrapure water to each membrane. Incubate at room
temperature for 2 min.
15. Spin the column at 11,000 Â g for 1 min and combine the
eluent from both columns in to one tube.
16. Analyze the purified dsDNA alongside the template using
DNA electrophoresis gel (1% (w/v) agarose) to estimate how
much of the dU-ssDNA has been converted in to
CCC-dsDNA. Load 1 μl of the reaction next to 1 μl of
dU-ssDNA. The CCC-dsDNA might show several bands:
Proteomic Peptide-Phage Display of PDZ Domains
49
PCR amplified oligonucleotide library using 0.2 units/μl,
37
C for 30 min, 85
C for 15 min, followed by flash cooling
on ice.
2. 5’Phosphorylate the DNA directly after flash cooling. Combine in a 1.5 ml micro centrifuge tube the library with 2 μl 10Â
TM buffer, 2 μl 10 mM ATP, and 1 μl 100 mM DTT. Add
water up to 20 μl.
3. Add 10 units of T4 polynucleotide kinase and incubate for 1 h
at 37
C. Use the phosphorylated sample directly for annealing.
4. To 10 μg of dU-ssDNA add 25 μl of 10Â TM buffer, 20 μl of
the phosphorylated oligonucleotides and adjust to a final volume of 250 μl.
5. Incubate at 90
C for 3 min, 50
C for 3 min and 20
C for
5 min.
6. Add 10 μl of 10 mM dNTP, 10 μl 10 mM ATP 15 μl 100 mM
DTT, 30 units T7 DNA polymerase, and 30 Weiss units of T4
DNA ligase to the annealed oligonucleotides.
7. Incubate the reaction for 16 h at 20
C. This will synthesize the
second strand and ligate it to form the heteroduplex
CCC-dsDNA.
8. Freeze-thaw the sample 3 times to inactivate the enzymes.
9. Add 5 μl Fast-digest SmaI to remove phagemid without insert
(see Note 3). Incubate for 30 min at 37
C.
10. Purify and desalt the CCC-dsDNA using QIAquick DNA
purification kit. Add 1 ml of QG buffer to the reaction and mix.
11. Load the sample to two spin columns placed in 2-ml microcentrifuge tubes. Centrifuge at 16,000 Â g for 1 min, discard
the flow through.
12. To each column add 750 μl of PE buffer and spin the sample at
16,000 Â g for 1 min, discard the flow through.
13. Centrifuge the columns an extra time at 16,000 Â g for 1 min
to remove excess PE buffer.
14. Transfer the column to a new 1.5 ml microcentrifuge tube, add
35 μl of ultrapure water to each membrane. Incubate at room
temperature for 2 min.
15. Spin the column at 11,000 Â g for 1 min and combine the
eluent from both columns in to one tube.
16. Analyze the purified dsDNA alongside the template using
DNA electrophoresis gel (1% (w/v) agarose) to estimate how
much of the dU-ssDNA has been converted in to
CCC-dsDNA. Load 1 μl of the reaction next to 1 μl of
dU-ssDNA. The CCC-dsDNA might show several bands:
Proteomic Peptide-Phage Display of PDZ Domains
49