PrbbBB:Oligo Annealing: Difference between revisions
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* Thermocycler | * Thermocycler | ||
* 10x annealing buffer, final (1x) concentration: 10mM Tris-HCl, 50mM NaCl, 1mM EDTA | * 10x annealing buffer, final (1x) concentration: 10mM Tris-HCl, 50mM NaCl, 1mM EDTA | ||
** closest match is NEB Buffer 2 (1x: 10mM TrisHCl, 50mM NaCl, 10mM MgCl<sub>2</sub>, 1mM DTT) | |||
==Procedure== | ==Procedure== | ||
Revision as of 04:28, 21 July 2010
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 [1]
- 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, 1mM EDTA
- closest match is NEB Buffer 2 (1x: 10mM TrisHCl, 50mM NaCl, 10mM MgCl2, 1mM DTT)
Procedure
- step 1
- 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
- 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 |
Contact
or instead, discuss this protocol.
