Steps	Problems	Possible Reason	Solution
9, 16, 25, 34, 43, 49, 64, 72, 77, 87	Band after SfiI digestions is too large or there is more tdan one band.	Multiple inserts	A) Repeat ligation witd less insert;or B) Digest library witd SpeI, clean vector by gel extraction, and religate;or C) use AhdI instead of SpeI to cut tde vector and insert; tdis will give a larger insert size (see Fig. 2 for approximate AhdI location).
8, 15, 24, 33, 42, 48, 50, 54, 86	Library size is too small	Low cell competency	A) Use a strain with higher competency potential;or B) make a fresh batch of competent cells;or C) do two (or more) simultaneous ligation reactions.
8, 15, 24, 33, 42, 48, 50, 54, 86	Background is too high	Insufficient digestion of vector	Re-digest vector with higher concentration of enzyme or for a longer period of time.
8, 15, 24, 33, 42, 48, 50, 54, 86	Background is too high	Insufficient dephosphorylation of vector	Clean vector by running on a 1.5% agarose gel and clean via “Freeze and Squeeze”69 or electroelution.