PrbbBB:Oligo Annealing

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(Materials)
(Procedure)
Line 21: Line 21:
==Procedure==
==Procedure==
-
# step 1
+
# mix:
-
# step 2
+
* 21 µl sterile ddH20
 +
* 3 µl sense oligo
 +
* 3 µl antisense oligo
 +
* 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==

Revision as of 08:31, 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
    • 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. . pmid:17293868. PubMed HubMed [Li2007]

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