PrbbBB:Oligo Annealing

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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:


 * Silver lab protocol (using water bath): Silver:_Oligonucleotide_Inserts
 * brief mentioning in Li & Elledge SLIC paper Li2007
 * Web protocol

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

Contact

 * Raik

or instead, discuss this protocol.