PrbbBB:Oligo Annealing

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* Thermocycler
* Thermocycler
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* some buffer
+
* 10x annealing buffer, final (1x) concentration: 10mM Tris-HCl, 50mM NaCl, 1mM EDTA
==Procedure==
==Procedure==

Revision as of 06:55, 21 July 2010

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Contents

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, 1mM EDTA

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. . pmid:17293868. PubMed HubMed [Li2007]

Contact

or instead, discuss this protocol.

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