TABLE 2.

Troubleshooting table.

Step	Problem	Possible reason	Solution
6, 28	Little DNA recovered or poor quality DNA prep	Slow growth of bacteria culture or poor DNA purification (multiple bands observed in agarose gel)	Grow large culture volumes: fUSE5, f88-4 and iPhage plasmids are considered low-copy number plasmids. Perform CsCl purification as described in STEP 8.
10, 28	Difficult to visualize the plasmid DNA band	Poor CsCl gradient separation	Make sure the proper amount of CsCl was used; changing the ultracentrifuge rotor could increase band definition/separation (e.g. try 70i.Ti).
19, 25, 43	Low ligation efficiency	Non-digested plasmids and/or insert; overexposure of DNA fragments to UV during the gel extraction procedure.	Ensure the complete digestion of the vector in an agarose gel by analyzing the DNA running pattern (see Step 34), the linear plasmid (cut) will appear to migrate slower (higher band) in the agarose gel than the same plasmid when circular (uncut – lower band); although the restriction enzymes used to create the iPhage vector and library generate incompatible ends, dephosphorylating the vector may help increase the ligation efficiency; increase restriction enzyme incubation time; use as little as possible of the vector plasmid (e.g 10 ng); purify ligation before electroporation as described in STEP 46; perform gel extraction by blind excision or use a 365 nm UV transilluminator instead of the usual 312 nm.
		Insufficient insert ligated to the vector	Optimize the vector:insert molar ratio as described in STEP 18.
34	Strong star activity in the iPhage DNA	Excess of SfiI endonuclease or prolonged incubation	Use fewer units (e.g. 50U) of SfiI or decrease incubation time (e.g. 2 h) to achieve complete digestion and avoid overdigestion.
57	Low iPhage titers	Old K91/kan E. coli	Check bacterial viability. Repeat infection of 1 ml of K91/kan E. coli bacteria with the new iPhage preparation for 1 h (10 ml at least), and grow in 500 ml for up to 16 h.
		Incorrect phage assembly	Sequence iPhage plasmid with the f88-4seq and pIIIseq primers sets (Table 1) to verify accurate assembly of the bifunctional iPhage vector.
91	Low or no protein recovered	Insufficient protein loaded in the column	Measure protein concentration by BCA or Bradford methods; optimize the amount of protein to be loaded on the column.
		Sample loading or elution conditions not favorable for protein purification	Optimize buffer pH; elution buffer is not optimal, increase concentration of bioactive peptide (e.g. 10 mM); alternatively, perform the elution step with 20 ml of Glycine buffer (pH 3.0) and recover 0.5 ml aliquots always monitoring the absorbance at 280 nm.
	Excess of nonspecific proteins	Improper column wash	Wash column more thoroughly before elution; wash columns with a more stringent second wash buffer (for example, with higher salt). The stringency may be increased by increasing the sodium chloride (NaCl) concentration to 0.5 M or even 1 M to reduce ionic and electrostatic attractions. Low levels of reducing agents (such as 1-2 mM DTT or BME) can help disrupt non-specific interactions mediated by disulfide bridges.
102	Too many colonies to count; no difference between fractions	Inaccurate phage titer	Check bacterial viability. Repeat infection of 1 ml of K91/kan E. coli bacteria with the new iPhage preparation for 1 h (10 ml at least), and grow in 500 ml for up to 16 h.
		Insufficient blocking or poor plate washing	Prepare fresh 1% BSA blocking solution and incubate plates at 4°C overnight. Add 0.05% Tween-20 (T) into PBS and wash the 96-well plate 20 times with 200 μl T-PBS.
		Excess of protein coated on the plate	Remeasure protein concentration by BCA or Bradford methods; optimize the amount of protein to be coated on the plate.
105	Difficulty identifying/extracting unique protein bands	Excess of receptor candidates in the fraction of interest	Perform a 2D gel analysis and remove protein spots of interest for mass spec analysis.
		Low protein concentration in the fraction of interest	Stain the gel with SYPRO or silver stain.
106	Keratin is the major protein identified by MS/MS	Sample contamination through environment rather than natural abundance	Use a biological safety cabinet or laminar flow hood; wear disposable lab coats; wear powder-free nitrile gloves; clean all surfaces with water and ethanol.