Difference between revisions of "PrbbBB:Oligo Annealing"

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(Procedure)
 
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* Thermocycler
 
* Thermocycler
* some buffer
+
* 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)
  
 
==Procedure==
 
==Procedure==
  
# step 1
+
mix (30µl total volume, 20µM final DNA concentration):
# step 2
+
* 15 µl sterile ddH20
 +
* 6 µl sense oligo 100µM
 +
* 6 µl antisense oligo 100µM
 +
* 3 µl NEB buffer 2 (10x)
 +
 
 +
incubate on thermocycler:
 +
* 2 min @ 98 C
 +
* 60 cycles:
 +
** 1 min, decreasing temperature by 1.3 C per cycle
 +
* cool to 4 C
  
 
==Notes==
 
==Notes==

Latest revision as of 05:35, 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

mix (30µl total volume, 20µM final DNA concentration):

  • 15 µl sterile ddH20
  • 6 µl sense oligo 100µM
  • 6 µl antisense oligo 100µM
  • 3 µl NEB buffer 2 (10x)

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 | PubMed ID:17293868 | HubMed [Li2007]

Contact

or instead, discuss this protocol.