Read spectrophotometrically at 450 nm in a microtiter plate
reader. Retrieve the results and visualize the ratios between the
A 450
target and A 450
control . The ratio should be higher than 2 (and
ideally more) for a selection to be considered successful.
3.8 Preparation
of Sample for NSG
Analysis
To analyze the results of the binding enriched phage pools, the
DNA sequences encoding the displayed peptides are sequenced
using NGS. To prepare phage pools for NGS, the DNA is amplified
and barcoded. Barcodes are unique stretches of nucleotides, which
identify the PCR product as coming from a specific phage pool.
Examples of barcodes and adaptors for Illumina sequencing can be
found elsewhere [15]. NGS facilities can typically provide advices
on barcoding strategies and provide support for demultiplexing (see
Note 6).
3.8.1 PCR Amplification
and Barcoding
1. Dissolve NGS grade primers (100 μM).
2. Dilute the primers to 5 μM and array them in a 96-well PCR
plate.
3. Dilute the out-phage pools 10Â in 2YT in a 96-well PCR plate.
The diluted phage supernatants are used as template in the
PCR reaction.
4. Prepare PCR samples by for each reaction mixing the following: 12.5 μl 2x Phusion Master Mix, 2.5 μl forward primer
(5 μM) and 2.5 μl reverse primer, 2.5 μl phage pool template
and 5 μl dH 2 O. Mix primers, template and dH 2 O first. Ensure
that the PCR machine is 98
C, then add Phusion Master Mix,
and start the PCR directly following the program in Table 1.
5. During the 30 min PCR, prepare a 2% agarose gel with GelRed
staining.
6. Remove the PCR plate from the machine directly after finished
program. Withdraw 1 μl of the reaction mixture for analysis
through agarose gel electrophoresis and freeze the plate at
À20
C.
7. Analyze the PCR products using 2% agarose gel electrophoresis
to verify the amplification of the template DNA.
3.8.2 Normalization
of PCR Products
A large number of samples (500-600 reactions) can be pooled into
one MiSeq run. The amount of DNA pooled from each PCR
reaction can be normalized through precipitation on magnetic
beads.
1. Thaw the PCR products.
2. Add 20 μl magnetic beads (Mag-Bind total Pure NGS) and
20 μl PCR product to a PCR plate well.
3. Mix by pipetting up and down 10 times.
Proteomic Peptide-Phage Display of PDZ Domains
57
reader. Retrieve the results and visualize the ratios between the
A 450
target and A 450
control . The ratio should be higher than 2 (and
ideally more) for a selection to be considered successful.
3.8 Preparation
of Sample for NSG
Analysis
To analyze the results of the binding enriched phage pools, the
DNA sequences encoding the displayed peptides are sequenced
using NGS. To prepare phage pools for NGS, the DNA is amplified
and barcoded. Barcodes are unique stretches of nucleotides, which
identify the PCR product as coming from a specific phage pool.
Examples of barcodes and adaptors for Illumina sequencing can be
found elsewhere [15]. NGS facilities can typically provide advices
on barcoding strategies and provide support for demultiplexing (see
Note 6).
3.8.1 PCR Amplification
and Barcoding
1. Dissolve NGS grade primers (100 μM).
2. Dilute the primers to 5 μM and array them in a 96-well PCR
plate.
3. Dilute the out-phage pools 10Â in 2YT in a 96-well PCR plate.
The diluted phage supernatants are used as template in the
PCR reaction.
4. Prepare PCR samples by for each reaction mixing the following: 12.5 μl 2x Phusion Master Mix, 2.5 μl forward primer
(5 μM) and 2.5 μl reverse primer, 2.5 μl phage pool template
and 5 μl dH 2 O. Mix primers, template and dH 2 O first. Ensure
that the PCR machine is 98
C, then add Phusion Master Mix,
and start the PCR directly following the program in Table 1.
5. During the 30 min PCR, prepare a 2% agarose gel with GelRed
staining.
6. Remove the PCR plate from the machine directly after finished
program. Withdraw 1 μl of the reaction mixture for analysis
through agarose gel electrophoresis and freeze the plate at
À20
C.
7. Analyze the PCR products using 2% agarose gel electrophoresis
to verify the amplification of the template DNA.
3.8.2 Normalization
of PCR Products
A large number of samples (500-600 reactions) can be pooled into
one MiSeq run. The amount of DNA pooled from each PCR
reaction can be normalized through precipitation on magnetic
beads.
1. Thaw the PCR products.
2. Add 20 μl magnetic beads (Mag-Bind total Pure NGS) and
20 μl PCR product to a PCR plate well.
3. Mix by pipetting up and down 10 times.
Proteomic Peptide-Phage Display of PDZ Domains
57