PvuI NEB#R0150

Protocol tips

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	The reaction mixture was purified with the Qiagen PCR Purification Kit and incubated with 5 units of Klenow Fragment (3′ exo−) (NEB) for 30 minutes at 37 °C in 1x NEBuffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9) with 240 μM dATP (Promega) in a 50 μL final volume. 10 mM Tris-HCl, pH 8.5 was added to a volume of 90 μL and the reaction was incubated for 20 minutes at 75 °C to inactivate the enzyme before cooling to 12 °C. 300 fmol of “adapter1/2”, barcoded according to enzyme concentration, or 6 pmol of “adapter1/2” for the PvuI digest, were added to the reaction mixture, along with 10 ul 10x NEB T4 DNA Ligase Reaction Buffer (500 mM Tris-HCl, 100 mM MgCl2, 100 mM dithiothreitol, 10 mM ATP).

Protocol tips

The reaction mixture was purified with the Qiagen PCR Purification Kit and incubated with 5 units of Klenow Fragment (3′ exo−) (NEB) for 30 minutes at 37 °C in 1x NEBuffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9) with 240 μM dATP (Promega) in a 50 μL final volume. 10 mM Tris-HCl, pH 8.5 was added to a volume of 90 μL and the reaction was incubated for 20 minutes at 75 °C to inactivate the enzyme before cooling to 12 °C. 300 fmol of “adapter1/2”, barcoded according to enzyme concentration, or 6 pmol of “adapter1/2” for the PvuI digest, were added to the reaction mixture, along with 10 ul 10x NEB T4 DNA Ligase Reaction Buffer (500 mM Tris-HCl, 100 mM MgCl2, 100 mM dithiothreitol, 10 mM ATP).

Publication protocol

Libraries of target sites were incorporated into double-stranded DNA by PCR with Taq DNA Polymerase (NEB) on a pUC19 starting template with primers “N5-PvuI” and “CCR5-224-N4,” “CCR5-224-N5,” “CCR5-224-N6,” “CCR5-224-N7,” “VF2468-N4,” “VF2468-N5,” “VF2468-N6,” or “VF2468-N7,” yielding an approximately 545-bp product with a PvuI restriction site adjacent to the library sequence, and purified with the Qiagen PCR Purification Kit. Library-encoding oligonucleotides were of the form 5′ backbone-PvuI site-NNNNNN-partially randomized half-site–N4–7–partially randomized half site-N-backbone 3′. The purified oligonucleotide mixture (approximately 10 μg) was blunted and phosphorylated with a mixture of 50 units of T4 Polynucleotide Kinase and 15 units of T4 DNA polymerase (NEBNext End Repair Enzyme Mix, NEB) in 1x NEBNext End Repair Reaction Buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP, 0.4 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP, 0.4 mM dTTP, pH 7.5) for 1.5 hours at room temperature. The blunt-ended and phosphorylated DNA was purified with the Qiagen PCR Purification Kit according to the manufacturer’s protocol, diluted to 10 ng/μL in NEB T4 DNA Ligase Buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP, pH 7.5) and circularized by ligation with 200 units of T4 DNA ligase (NEB) for 15.5 hours at room temperature. Circular monomers were gel purified on 1% TAE-Agarose gels. 70 ng of circular monomer was used as a substrate for rolling-circle amplification at 30 °C for 20 hours in a 100 μL reaction using the Illustra TempliPhi 100 Amplification Kit (GE Healthcare). Reactions were stopped by incubation at 65 °C for 10 minutes. Target site libraries were quantified with the Quant-iT PicoGreen dsDNA Reagent (Invitrogen). Libraries with N4, N5, N6, and N7 spacer sequences between partially randomized half-sites were pooled in equimolar concentrations for both CCR5-224 and VF2468.

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