PrbbBB:Oligo Annealing

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Overview

Short to medium length (up to 120 bp) fragments of dsDNA can be generated directly from two complementary single-stranded oligonucleotides. Custom overhangs for ligation or In-Fusion can be introduced as well, simply by having some non-complementary ends. The two single-stranded Oligos then need to be annealed into a double-stranded fragment.

See also:

• Silver lab protocol (using water bath): Silver:_Oligonucleotide_Inserts
• brief mentioning in Li & Elledge SLIC paper [1]
• Web protocol

The idea is to heat up the mixture to close to 100 C and let it slowly cool down to room temperature over the course of one hour.

Materials

• Thermocycler
• 10x annealing buffer, final (1x) concentration: 10mM Tris-HCl, 50mM NaCl
  • closest match is NEB Buffer 2 (1x: 10mM TrisHCl, 50mM NaCl, 10mM MgCl₂, 1mM DTT)

Procedure

1. mix:

• 21 µl sterile ddH20
• 3 µl sense oligo
• 3 µl antisense oligo
• 3 µl NEB buffer 2 (10x)

1. incubate on thermocycler

• 2 min @ 98 C
• 60 cycles:
  • 1 min, decreasing temperature by 1.3 C per cycle
• cool to 4 C

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

raik: We are just starting to use this protocol -- share your experience!

References

1. Li MZ and Elledge SJ. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods. 2007 Mar;4(3):251-6. DOI:10.1038/nmeth1010 |

Contact

• Raik

or instead, discuss this protocol.