17. Purify the extracted PCR product using a gel purification kit.
Dissolve the gel completely by an extended incubation in
denaturing buffer QG at room temperature. Do not vortex,
but gently turn the tube up and down. Elute in TE buffer.
18. Quantify the dsDNA concentration using PicoGreen, as
described in Subheading 3.1.
19. Provide the required amount to the sequencing facility following the instructions.
20. Results are typically returned in FASTQ format. The results are
then demultiplexed and matched to the library design as
described elsewhere [16].
4 Notes
1. We quantify the purified PCR product using PicoGreen since
Nanodrop quantification is unreliable for the determination,
and tend to overestimate the concentration.
2. Pick several colonies to make sure that at least one will grow
efficiently.
3. We have inserted a SmaI restriction site in our phagemid vector
in between the annealing sites of the library oligonucleotides,
and ensured that there is no SmaI site in the designed oligonucleotide pool. This step can be omitted.
4. The phage library can be used directly, or reamplified in presence of 0.3 mM IPTG.
5. If there is growth in the other cultures then discard the current
cultures and start a new one).
6. The binding enriched phages can also be analyzed through
clonal phage ELISA and Sanger sequencing, as described
elsewhere [11].
7. Pay attention not to transfer any beads at this stage. In case
there are still some beads in the solution after transfer, then
pellet the magnetic beads using a magnetic stand, and transfer
the solution to a new vial.
Acknowledgments
This work was supported by the European Union’s Horizon 2020
research and innovation program under the Marie SklodowskaCurie grant agreement no. 675341.
Proteomic Peptide-Phage Display of PDZ Domains
59
Dissolve the gel completely by an extended incubation in
denaturing buffer QG at room temperature. Do not vortex,
but gently turn the tube up and down. Elute in TE buffer.
18. Quantify the dsDNA concentration using PicoGreen, as
described in Subheading 3.1.
19. Provide the required amount to the sequencing facility following the instructions.
20. Results are typically returned in FASTQ format. The results are
then demultiplexed and matched to the library design as
described elsewhere [16].
4 Notes
1. We quantify the purified PCR product using PicoGreen since
Nanodrop quantification is unreliable for the determination,
and tend to overestimate the concentration.
2. Pick several colonies to make sure that at least one will grow
efficiently.
3. We have inserted a SmaI restriction site in our phagemid vector
in between the annealing sites of the library oligonucleotides,
and ensured that there is no SmaI site in the designed oligonucleotide pool. This step can be omitted.
4. The phage library can be used directly, or reamplified in presence of 0.3 mM IPTG.
5. If there is growth in the other cultures then discard the current
cultures and start a new one).
6. The binding enriched phages can also be analyzed through
clonal phage ELISA and Sanger sequencing, as described
elsewhere [11].
7. Pay attention not to transfer any beads at this stage. In case
there are still some beads in the solution after transfer, then
pellet the magnetic beads using a magnetic stand, and transfer
the solution to a new vial.
Acknowledgments
This work was supported by the European Union’s Horizon 2020
research and innovation program under the Marie SklodowskaCurie grant agreement no. 675341.
Proteomic Peptide-Phage Display of PDZ Domains
59