Difference between revisions of "PrbbBB:Oligo Annealing"

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(Materials)
(Materials)
Line 16: Line 16:
  
 
* Thermocycler
 
* Thermocycler
* 10x annealing buffer, final (1x) concentration: 10mM Tris-HCl, 50mM NaCl, 1mM EDTA
+
* 10x annealing buffer, final (1x) concentration: 10mM Tris-HCl, 50mM NaCl
 
** closest match is NEB Buffer 2 (1x: 10mM TrisHCl, 50mM NaCl, 10mM MgCl<sub>2</sub>, 1mM DTT)
 
** closest match is NEB Buffer 2 (1x: 10mM TrisHCl, 50mM NaCl, 10mM MgCl<sub>2</sub>, 1mM DTT)
  

Revision as of 03:28, 21 July 2010

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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:

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. step 1
  2. step 2

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 | PubMed ID:17293868 | HubMed [Li2007]

Contact

or instead, discuss this protocol.