Creating sgRNA library using ssDNA bridging

Creating sgRNA library using ssDNA bridging

Efficient Cloning with NEBuilder HiFi and ssDNA Oligos

Save time and resources in your lab by using NEBuilder HiFi DNA Assembly with single-stranded oligos (ssDNA). This guide explains how to bypass expensive dsDNA synthesis services for your cloning projects.

Two Powerful ssDNA Strategies

Strategy A. Multiple Oligo Assembly: Overlap several short ssDNAs (60nt each) to form a long, functional DNA fragment.

Strategy B. ssDNA Bridging: Use a single ssDNA oligo to bridge gaps between linearized vector ends, ideal for sgRNA-Cas9 constructs.

Step-by-Step Protocol

1. Oligo Design & Annealing

Design each oligo with a 30nt overlap to its neighbor. 5' phosphorylation is NOT required. Prepare in 1X NEBuffer 2:

  • Step 1: Mix 100uM oligos (Final concentration ~0.2uM)
  • Step 2: Heat at 100°C for 3 minutes
  • Step 3: Cool slowly at room temperature for 20 minutes

2. Assembly Reaction Setup

Component Volume Note
Annealed/Diluted Oligos 5 ul 0.2 uM stock
Linearized Vector 1 ul 0.02 pmol/ul
Nuclease-free Water 4 ul -
2X NEBuilder HiFi Mix 10 ul Master Mix
Total 20 ul -
💡 Pro-Tip: Transformation Success

Incubate the reaction at 50°C for 60 minutes. For maximum efficiency, transform 2ul into NEB 5-alpha competent cells following the standard heat-shock protocol.

Keywords: NEBuilder HiFi, ssDNA Oligo, DNA Assembly, Cloning Protocol, sgRNA-Cas9, Nicked dsDNA, Oligo Design, NEB E2621

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