3.2 Quantification
of the PCR Product
(See Note 1)
1. Dilute 10Â TE buffer in MilliQ water to 1Â.
2. Dilute PicoGreen dye 1:400 in TE buffer.
3. Prepare duplicate of 8 steps two-times serial dilution of lambda
phage dsDNA standard stock (100 μg/ml dsDNA).
4. Add 25 μl of the dye to 16 wells in a 96-well plate compatible
with a qPCR instrument (e.g., white well hard-shell PCR plates
96-well, thin wall). Add 1 μl of the serial dilution to each well.
5. Prepare 1:2, 1:4, and 1:8 dilutions of your PCR reaction.
6. Mix 1 μl of the dilution with 25 μl dye in separate wells.
7. Spin the plates at 100 Â g to ensure that the solution is at the
bottom of the wells.
8. Run a single cycle 20
C 1 min qPCR with a readout of SYBR or
fluorescein settings: excitation ~480 nm, emission 520 nm.
9. Generate a standard curve using the duplicate dilutions that are
in the linear range.
10. Determine the DNA concentration of the diluted PCR reaction from the standard curve.
3.3 Purification
of dU-ssDNA
Phagemid
This protocol will yield dU-ssDNA in amounts exceeding 20 μg,
which is enough for production of 2-4 ProP-PD libraries depending on library diversity, using a modified version of the QIAprep
spin kit described previously [11].
1. Transform your phagemid into CJ360 (or alternative dut
À /
ung
À strain) and plate it on an LB/carb plate. Incubate overnight at 37
C.
2. Inoculate a single CJ360 colony into 1 ml of 2YT medium
supplemented with M13KO7 helper phage (10
10 pfu/ml),
100 μg/ml carbenicillin (final concentration) and 34 μg/ml
chloramphenicol to maintain the phagemid and the epitope for
the F
0 episome of E. coli CJ236. Incubate the culture for 2 h in
37
C with shaking at 200 rpm (see Note 2).
3. Add 25 μg/ml kanamycin (final concentration) to select for
bacteria coinfected with M13KO7 helper phage. Shake at
200 rpm for 6 h at 37
C.
4. Transfer the bacterial culture into 30 ml 2YT/carb/kan/uridine medium and incubate at 37
C for 20 h with shaking at
200 rpm.
5. Pellet the bacteria by centrifugation for 10 min at 12,000 Â g
using a Beckman JA14.50 rotor or equivalent. Transfer the
supernatant into a new tube containing 1/5 the final volume
of PEG/NaCl. Incubate on ice for 5 min.
Proteomic Peptide-Phage Display of PDZ Domains
47
of the PCR Product
(See Note 1)
1. Dilute 10Â TE buffer in MilliQ water to 1Â.
2. Dilute PicoGreen dye 1:400 in TE buffer.
3. Prepare duplicate of 8 steps two-times serial dilution of lambda
phage dsDNA standard stock (100 μg/ml dsDNA).
4. Add 25 μl of the dye to 16 wells in a 96-well plate compatible
with a qPCR instrument (e.g., white well hard-shell PCR plates
96-well, thin wall). Add 1 μl of the serial dilution to each well.
5. Prepare 1:2, 1:4, and 1:8 dilutions of your PCR reaction.
6. Mix 1 μl of the dilution with 25 μl dye in separate wells.
7. Spin the plates at 100 Â g to ensure that the solution is at the
bottom of the wells.
8. Run a single cycle 20
C 1 min qPCR with a readout of SYBR or
fluorescein settings: excitation ~480 nm, emission 520 nm.
9. Generate a standard curve using the duplicate dilutions that are
in the linear range.
10. Determine the DNA concentration of the diluted PCR reaction from the standard curve.
3.3 Purification
of dU-ssDNA
Phagemid
This protocol will yield dU-ssDNA in amounts exceeding 20 μg,
which is enough for production of 2-4 ProP-PD libraries depending on library diversity, using a modified version of the QIAprep
spin kit described previously [11].
1. Transform your phagemid into CJ360 (or alternative dut
À /
ung
À strain) and plate it on an LB/carb plate. Incubate overnight at 37
C.
2. Inoculate a single CJ360 colony into 1 ml of 2YT medium
supplemented with M13KO7 helper phage (10
10 pfu/ml),
100 μg/ml carbenicillin (final concentration) and 34 μg/ml
chloramphenicol to maintain the phagemid and the epitope for
the F
0 episome of E. coli CJ236. Incubate the culture for 2 h in
37
C with shaking at 200 rpm (see Note 2).
3. Add 25 μg/ml kanamycin (final concentration) to select for
bacteria coinfected with M13KO7 helper phage. Shake at
200 rpm for 6 h at 37
C.
4. Transfer the bacterial culture into 30 ml 2YT/carb/kan/uridine medium and incubate at 37
C for 20 h with shaking at
200 rpm.
5. Pellet the bacteria by centrifugation for 10 min at 12,000 Â g
using a Beckman JA14.50 rotor or equivalent. Transfer the
supernatant into a new tube containing 1/5 the final volume
of PEG/NaCl. Incubate on ice for 5 min.
Proteomic Peptide-Phage Display of PDZ Domains
47