Team:Northwestern/Notebook/Protocols/Oligo Annealing

From 2011.igem.org

(Difference between revisions)
 
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'''Overview'''
'''Overview'''
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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. 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.  
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. 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.  
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'''Materials'''
'''Materials'''
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* Thermocycler
* Thermocycler
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'''Procedure'''
'''Procedure'''
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Mix (30µl total volume, 20µM final DNA concentration):
Mix (30µl total volume, 20µM final DNA concentration):
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* 6 µl antisense oligo 100µM
* 6 µl antisense oligo 100µM
* 3 µl NEB buffer 2 (10x)
* 3 µl NEB buffer 2 (10x)
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-
 
Incubate on thermocycler:
Incubate on thermocycler:
* 2 min @ 98C
* 2 min @ 98C

Latest revision as of 15:34, 25 August 2011

RETURN TO IGEM 2010


Oligo Annealing


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


Adapted from [http://openwetware.org/wiki/PrbbBB:Oligo_Annealing OpenWetWare]

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