2.8 Preparation
and Quantification
of Sample
for Next-Generation
Sequencing (NGS)
1. Phusion high fidelity PCR master mix with HF buffer.
2. NGS grade PCR primers with barcodes.
3. Mag-Bind total Pure NGS.
4. EB Buffer: provided by the kit manufacturer.
5. 10Â TE buffer
6. QG buffer.
7. 70% ethanol
8. GelRed.
9. Agarose.
10. 10Â TBE
11. Quant-iT PicoGreen dsDNA assay kit.
3 Methods
3.1 PCR
Amplification of a
Commercial Custom
Oligo Pools
Custom designed oligonucleotide libraries can be obtained from
commercial providers. The details of the design of the oligonucleotide libraries will depend on the research questions, which is outside
the scope of this protocol. In general terms, we retrieve the peptide
sequences of interest from a reliable source (e.g., Uniprot),
reversely translate the regions of interest, and add on flanking
oligonucleotide stretches that are complementary to the phagemid
vector. For the C-terminal display on the M13 phage we construct
the library using a phagemid that encodes an engineered version of
the major coat protein P8 engineered by the Sidhu lab [12]. To
remove 5
0 and 3
0 adaptor sequences we typically amplify the
obtained oligonucleotide library before use through the following
steps.
1. Prepare 4–8 PCR reactions mixing 5 μl of 5 μM forward and
reverse primers each, 25 μl of Phusion high fidelity PCR master
mix with Phusion HF buffer, 1 μl of the custom oligonucleotide pool and 14 μl H 2 O.
2. Run a PCR for 18 cycles with denaturation 15 s at 98
C,
annealing at 56
C for 15 s, and elongation at 72
C for 10 s.
A rather low number of PCR cycles (18–20) is used to reduce
the PCR bias.
3. Verify the product by electrophoresis, using a 2% agarose gel
with GelRed staining and loading 1 μl of each PCR reaction.
4. Pool the four best reactions and clean them up using Nucleotide Removal Kit. Elute in 30 μl of elution buffer. Note that the
PCR primers are most likely not removed by this, but they will
be removed at a later stage.
46
Susanne Lu ¨ chow et al.
and Quantification
of Sample
for Next-Generation
Sequencing (NGS)
1. Phusion high fidelity PCR master mix with HF buffer.
2. NGS grade PCR primers with barcodes.
3. Mag-Bind total Pure NGS.
4. EB Buffer: provided by the kit manufacturer.
5. 10Â TE buffer
6. QG buffer.
7. 70% ethanol
8. GelRed.
9. Agarose.
10. 10Â TBE
11. Quant-iT PicoGreen dsDNA assay kit.
3 Methods
3.1 PCR
Amplification of a
Commercial Custom
Oligo Pools
Custom designed oligonucleotide libraries can be obtained from
commercial providers. The details of the design of the oligonucleotide libraries will depend on the research questions, which is outside
the scope of this protocol. In general terms, we retrieve the peptide
sequences of interest from a reliable source (e.g., Uniprot),
reversely translate the regions of interest, and add on flanking
oligonucleotide stretches that are complementary to the phagemid
vector. For the C-terminal display on the M13 phage we construct
the library using a phagemid that encodes an engineered version of
the major coat protein P8 engineered by the Sidhu lab [12]. To
remove 5
0 and 3
0 adaptor sequences we typically amplify the
obtained oligonucleotide library before use through the following
steps.
1. Prepare 4–8 PCR reactions mixing 5 μl of 5 μM forward and
reverse primers each, 25 μl of Phusion high fidelity PCR master
mix with Phusion HF buffer, 1 μl of the custom oligonucleotide pool and 14 μl H 2 O.
2. Run a PCR for 18 cycles with denaturation 15 s at 98
C,
annealing at 56
C for 15 s, and elongation at 72
C for 10 s.
A rather low number of PCR cycles (18–20) is used to reduce
the PCR bias.
3. Verify the product by electrophoresis, using a 2% agarose gel
with GelRed staining and loading 1 μl of each PCR reaction.
4. Pool the four best reactions and clean them up using Nucleotide Removal Kit. Elute in 30 μl of elution buffer. Note that the
PCR primers are most likely not removed by this, but they will
be removed at a later stage.
46
Susanne Lu ¨ chow et al.