Protocol for assembling annealed DNA oligonucleotides and a double-stranded DNA vector using NEBuilder HiFi DNA Assembly (NEB #E2621)
The following method allows you to anneal short overlapping DNA oligos (generally 60 nucleotides (nt) each) to assemble a longer double-stranded DNA (dsDNA) fragment. The annealed nicked dsDNA fragment can then be combined and assembled with a linearized vector fragment. This annealed oligo protocol provides an alternative to short, synthesized dsDNA, such as gBlocks™.
This protocol is recommended for the assembly of the following types of DNA fragments:
- Annealed short DNA oligos forming a nicked dsDNA fragment
- dsDNA vector linearized by PCR or restriction digest
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Annealed DNA Oligo Design
Short, annealed ssDNA oligos (60 nt each) should be designed with 30 nt overlaps with adjacent complementary oligos. When annealed, the overlapping oligos will form a nicked dsDNA fragment with no gaps, and ssDNA vector overlaps at each end. Please note that DNA oligos with 5’ phosphates are not required.
DNA Quantities
This protocol uses a 1:50 (vector:insert) molar ratio with 0.02 picomoles of vector and 1 picomole of annealed oligos.
Protocol
- Prepare oligos for annealing by adding 1 ul of each oligo (100 μM stock) to a final concentration of 0.2 μM (0.2 pmol/μl) using 1X NEBuffer r2.1*. This can be done by combining 1 µl of each 100 μM oligo stock in a single tube with an appropriate volume of buffer for a total volume of 500 μl.
- Heat the oligo mixture solution at 100°C for 3 min and allow to cool at room temperature for 20 min. The annealed oligos are ready to assemble.
- Set-up the following reaction on ice:
- Incubate the reaction at 50°C in a thermocycler for 60 min. Transform 2 μl of assembled mix into 50 μl of NEB 5-alpha Competent E. coli (High Efficiency) (NEB #C2987) following the recommended protocol.
Component | 500 µl Volume | Final Conc. Or Amount |
---|---|---|
Overlapping Oligos (100 μM stock concentration) | 1 μl of each oligo, for a total of X μl | 0.2 μM (0.2 pmol/μl) of each oligo |
1X NEBuffer r2.1* | Buffer volume = (500 μl - X μl) | 1X |
*Note: you can also use TE buffer (10 mM Tris, 0.1 mM EDTA; pH 8.0) supplemented with 50 mM NaCl as an annealing buffer
Component | 20 µl Reaction | Final Conc. Or Amount |
---|---|---|
Annealed Oligo Mixture (0.2 pmol/μl) |
5 μl | 1 pmol (1:50 vector:insert ratio) |
Linearized Vector (0.02 pmol/μl)** | 1 μl | 0.02 pmol |
Nuclease-free Water | 4 μl | |
NEBuilder HiFi DNA Assembly Master Mix 2X | 10 μl | 1X |
** NEBioCalculator (NEBioCalculator.neb.com) can help with DNA mass to molar quantity conversions for both ssDNA and dsDNA.
Note: If you are working with large plasmids >10 kb in size we recommend NEB® 10-beta Competent E. coli (High Efficiency) (NEB #C3019H). If your plasmid or insert contain repetitive sequences, we recommend NEB® Stable Competent E. coli (High Efficiency) (NEB #C3040H).