Mar 14, 2022

Public workspacePhage Display Library Prep Method

DOI
dx.doi.org/10.17504/protocols.io.rm7vz3945gx1/v1
  • 1Chan Zuckerberg Biohub, UCSF;
  • 2University of California, San Francisco;
  • 3University of California San Francisco
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DOI: dx.doi.org/10.17504/protocols.io.rm7vz3945gx1/v1
Protocol Citation: Sabrina A Mann, Lillian Khan, Sara Vazquez, Caleigh Mandel-Brehm, Joseph Derisi 2022. Phage Display Library Prep Method. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz3945gx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: July 01, 2021
Last Modified: March 14, 2022
Protocol Integer ID: 51226
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Abstract
This protocol was designed to be used after the Scaled High Throughput Vacuum PhIP Protocol or the Scaled Moderate Throughput Multichannel PhIP Protocol. The immunoprecipitated phage targets are prepared for sequencing through a two PCR amplification rounds which first amplify the peptide target and second, add on UMIs.
Materials
ReagentPhusion High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0530L
ReagentAgencourt AmPure XP beadsCatalog #A63880
ReagentQubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854
Reagent Bioanalyzer chips and reagents (DNA High Sensitivity kit)Agilent Technologies

Protocol materials
Reagent Bioanalyzer chips and reagents (DNA High Sensitivity kit)Agilent Technologies
Materials, Step 11
ReagentPhusion High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0530L
In Materials and 2 steps
ReagentAgencourt AmPure XP beadsCatalog #A63880
Materials, Step 10
ReagentQubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854
Materials, Step 11
Lysis
Lysis
Dilute phage lysis product in water at a 1:4 ratio. Phage lysis should be used either immediately after IP or within 1 week of being stored at Temperature4 °C .

  • NOTE: we have done several tests and noticed that diluting the phage lysate 1:4 in water results in the most product at the end. Other dilutions ranging from 1:2 to 1:16 also worked. Using undiluted lysate also works, however there is the possibility that too much input can inhibit the PCR.
Heat lysate in preparation for PCR at Temperature70 °C for Duration00:15:00 (can be done on the PCR machine if aliquoted into PCR tubes or plates). Keep on ice afterwards.
15m
PCR 1- Amplifying Insert
PCR 1- Amplifying Insert
15m
15m
ReagentPhusion High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0530L
  1. Add the following reagents. Reaction volumes of Amount50 µL , Amount25 µL or Amount12.5 µL all work.
  2. Multiply the number of samples by either the Amount50 µL , Amount25 µL or Amount12.5 µL reaction coefficients to get the total volumes of each reagent to add to the master mix.
  3. Make master mix and aliquot into a new PCR plate.
  4. Add pre-heated phage lysis to PCR I master mix.
ABCD
COMPONENT 50uL REACTION 25uL REACTION 12.5uL REACTION
Nuclease-free water 34.5 16.25 8.125
5X Phusion HF or GC Buffer 10 5 2.5
10 mM dNTPs 1 0.5 0.25
10 uM Forward Primer 1 0.5 0.25
10 uM Reverse Primer 1 0.5 0.25
Polymerase 0.5 0.25 0.125
Template DNA (lysis) 2 2 1
Total amount of Master Mix 48 23 11.5
Program PCR Machine for the following steps:
ABC
STEP TEMP TIME
Initial Denaturation 98°C 30 seconds
13-16 Cycles 98°C 5 seconds
70°C 20 seconds
72°C 15 seconds
Final Extension 72°C 2 minutes
Hold 10°C

PCR 2- Adding on UMI
PCR 2- Adding on UMI
ReagentPhusion High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0530L

  1. Multiply number of samples by either the Amount50 µL , Amount25 µL or Amount12.5 µL reaction number coefficients to get total volume of each reagent to add to master mix.
  2. Combine the following reagents (except barcode primers and PCR I product) to make PCR II master mix, then aliquot master mix volume into new PCR plate
  3. Next, add barcode primers and PCR I product, making sure to keep track of which barcode IDs corresponds to which sample well.
ABCD
COMPONENT 50uL REACTION 25uL REACTION 12.5uL REACTION
Nuclease-free water 33.5 16.25 8.125
5X Phusion HF or GC Buffer 10 5 2.5
10 mM dNTPs 1 0.5 0.25
Polymerase 0.5 0.25 0.125
5 uM Forward and Reverse Primer Barcodes 4 2 1
Template DNA (product of PCR I) 1 1 0.5
Total amount of Master Mix 46 22 11

Program the PCR machine for the following steps:

ABC
STEP TEMP TIME
Initial Denaturation 98°C 30 seconds
5 Cycles 98°C 5 seconds
70°C 20 seconds
72°C 15 seconds
Final Extension 72°C 2 minutes
Hold 10°C
Pool and Bead Clean
Pool and Bead Clean
Amplified samples are now uniquely labeled and are very similar in concentration and library size, so equal volume of each sample can be pooled together. If there is concern that samples are dissimilar, skip this step, bead clean, BioAnalyze and Qubit each sample individually. However, in our experience it is easier to pool first and then bead clean everything together prior to the QC check via BioAnalyzer and Qubit.

If pooling, it is recommend to pool anywhere between Amount5 µL -Amount10 µL of each sample together by using a multichannel and combining all of the rows into a reservoir.

  • NOTE: It is also recommended to pool each plate separately from other plates. This allows us to protect individual plates from phage contamination.
ReagentAgencourt AmPure XP beadsCatalog #A63880

*Allow beads to sit in TemperatureRoom temperature for Duration00:30:00 prior
*Ratio of beads changes based on application and library size.

  1. Determine the total volume of the pool. (i.e. 100uL)
  2. Use Agencourt AmPure XP Beads 0.9x ratio of beads-to-total volume of sample. Prepare 80% EtOH.
  3. Add 0.9x (i.e. 90uL) beads of room temperature AmPure Beads to pool. Mix well by pipetting up and down gently.
  4. Pulse spin the tube but do not spin down beads. Incubate for Duration00:05:00 at TemperatureRoom temperature .
  5. Place samples on magnetic rack and incubate for Duration00:05:00 on the rack.
  6. Remove supernatant without disturbing the beads.
  7. Add 2x original volume of 80% EtOH or enough to submerge bead pellet while on the magnetic rack. Incubate at TemperatureRoom temperature for Duration00:00:30 then remove the supernatant.
  8. Repeat EtOH wash (step 7) for a total of 2 times.
  9. Air dry the beads for Duration00:05:00 while on the magnetic rack.
  10. Remove tube from magnetic rack. Elute DNA from beads in desired volume of 0.1x TE Buffer, 10mM Tris-HCl, or Nuclease free water plus Amount3 µL of dead volume.
  11. Vortex to mix. Pulse spin tubes and incubate for Duration00:02:00 at TemperatureRoom temperature off the magnetic rack.
  12. Place on magnetic rack until solution is clear and bead pellet has formed ~ Duration00:05:00 .
  13. Remove desired volume of eluant and transfer to a clean nuclease-free PCR tube. Do not disturbe the bead pellet.
52m 30s
Quantify and Qualify Library
Quantify and Qualify Library
ReagentQubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854
Reagent Bioanalyzer chips and reagents (DNA High Sensitivity kit)Agilent Technologies

  1. Qubit the samples for accurate concentration. BioAnalyzer also gives the concentration of sample but in practice this number not as accurate as the Qubit result.The Qubit is our gold standard for confirming DNA concentration for dilution prior to using the BioAnalyzer as to not overload it.
  2. BioAnalyze or TapeStation library pools.
  3. If the library size looks as expected, pool separate plate pools together at equal concentration or at desired ratio for read depth. Final libraries can be stored at Temperature4 °C for a few weeks. For longterm storage, samples should be kept at Temperature-20 °C .

Expected result of library on a BioAnalyzer, it is normal to see these two peaks, the first peak should be smaller than the second. Make sure to confirm that the size of the main peak is the predicted size based on insert and primer scheme.