PrbbBB:Oligo Annealing: Difference between revisions
(New page: Back to all protocols ==Overview== Short to medium length (up to 120 bp) fragments of dsDNA can be generated directly from two complementary single-stranded oligonu...) |
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* Silver lab protocol (using water bath): [[Silver:_Oligonucleotide_Inserts]] | * Silver lab protocol (using water bath): [[Silver:_Oligonucleotide_Inserts]] | ||
* brief mentioning in Li & Elledge SLIC paper <cite>Li2007</cite> | * brief mentioning in Li & Elledge SLIC paper <cite>Li2007</cite> | ||
* [http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1282 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. | 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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* Thermocycler | * Thermocycler | ||
* | * 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== | ||
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== | ==Notes== | ||
Latest revision as of 05:35, 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
- 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
- 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.
