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  • Phage Display Troubleshooting Guide

    Problem area Problem Possible Cause Solution
    Titering M13 No plaques F’ of E. coli ER2738 lost
    • Re-streak ER2738 LB/Tet plate
    • check Tet stock
    Poor bacterial lawn growth
    • Plate incubator must be 37˚C
    • Agarose top must be £ 54 ˚C
    Inconsistent titer results and/or blue tinged (confluent) plates Phage solutions not dilute enough
    • Use 109-1011 dilutions for amplified stocks
    • Elution titers will vary widely
    Cross-contamination during dilution steps or plating
    • Use aerosol barrier pipet tips, change at each step
    ER2738 liquid culture or plate contaminated with phage
    • Re-streak ER2738 on LB/Tet plate
    • Include bacteria alone negative control plate for titer
    Smeared blue coloring on plates Excess moisture in agarose top or IPTG/Xgal plates
    • Allow plates to sit at RT or 37˚C for ~8hrs to dry
    • Difficult to eliminate totally
    Amplification No phage pellet (failed or low yield amplification) E. coli have lost F’
    • Re-streak ER2738 LB/Tet plate
    • Check Tet stock
    Cells overgrown prior to addition of phage
    • Use 1:100 dilution of overnight or add phage at 0.01-0.05 OD600 of same day culture
    Failed PEG/NaCl precipitation
    • Titer supernatant to check
    • Use PEG-8000, PEG/NaCl stock should be homogeneous
    Poor culture conditions
    • Use less than or equal to 5 g NaCl per L LB media
    • Aeration must be good (e.g. 250 mL flask for 20 mL culture)
    Clear plaques predominate after amplifying elution pool Environmental contamination from contaminated media, buffers, target or work area
    • Re-make solutions and autoclave
    • wipe down work area with dilute ethanol or bleach
    • check ER2738 plate for contamination
    Too many rounds of selection (>4-5)
    • Modify panning protocol to make binding conditions more stringent
    • Refine elution method
    After Selections
    Sequencing phage clones High A280/A260 for purified ssDNA Protein contamination, A280/A260 of 3-8 is typical from NaI ssDNA protocol
    • Sequencing reactions should still work
    • For cleaner templates include PCI extractions or use miniprep kit for ss or dsDNA
    Poor or no sequencing data Residual NaI interfering with reactions
    • Try diluting templates 3-fold
    If only, in random region it is a mixture of templates
    • Pick only well-spaced plaques from plates 1-2 days old maximum
    Multiple bands on agarose gel, unexpected sizes Single- and double- stranded DNA mixture, ssDNA will not migrate with dsDNA of same size
    • Multiple bands is typical -use M13mp18 (#N4040) as control
    • It is difficult to separate completely ss from ds phage DNA; and not necessary for this application
    My sequencing facility requires more template or primer than in NEB’s protocol
    • We recommend following instructions from the facility. However, we typically use 1pmol primer and 0.5-1 ug ss template per reaction
    I cannot find random region (displayed peptide codons) in my data Did not look at reverse compliment of raw data from ‑96seq primer
    • Take reverse compliment, search for KpnI and EagI sites. Compare what is in between these with Fig 5 in manual
    Clone has no insert or multiple inserts
    • Usually these are nonspecific binders; phage ELISA will confirm
    I am out of sequencing primer solution that came in my kit Kits provide enough primer to sequence ~100 clones
    • NEB does not sell primers separately anymore
    • Order oligo from your preferred supplier (-96seqp: 5’-CCCTCATAGTTAGCGTAACG)
    Post-panning analysis No consensus sequence after 3 rounds Conformational motif, single clones with specificity
    • Carryout phage ELISA or other binding assay
    • Carryout 4th round of selection
    No ELISA signal despite consensus sequence Selected clones are weak binders
    • Phage ELISA binding is not sensitive enough to detect weaker (mM) interactions
    No consensus and no hits from phage ELISA Selected phage are weak binders, target unrelated selections or no phage were bound
    • Consider modifying panning strategy to eliminate target unrelated binding activity
    Synthetic peptides do not bind despite phage particle binding Phage display 5-copies of each peptide
    • Due to avidity affects, multimers may be required for affinity to the target
    Synthetic peptide design is dissimilar to phage
    • C-terminus should be not charged; consider amidation
    • Also, consider using GGGS linker