DNA Synthesis from Oligos: Difference between revisions
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== Notes == | == Notes == | ||
This works for relatively | This works well for relatively for short fragments (300-500 bp). For longer sequences, use [[PCR Overlap Extension]] of the fragments. | ||
== References == | == References == | ||
Rouillard et al. Gene2Olig: oligonucleotide design for ''in vitro'' gene syntehsis. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15215375] | Rouillard et al. Gene2Olig: oligonucleotide design for ''in vitro'' gene syntehsis. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15215375] | ||
Revision as of 11:41, 29 May 2006
Overview
Despite claims by Synthesis companies for cheap gene/DNA synthesis, there are times when manual synthesis is necessary. This can be done relatively easily by ordering the necessary oligos of both strands, and a bit of ligation cycling.
Procedure
- Design oligos for your gene, of both strands. Either make them fixed 30-mers, with 15-mer overlaps, or use the software by Rouillard et al. referenced below.
- Dilute the oligos to 100uM.
- Kinase the oligos.
- Ligation cycle:
- For Thermostable Taq ligase, cycle between 95C (denature), 45-55C (anneal), and 65C (ligate). Do many as necessary (10-30).
- For T4 ligase, cycle between 95C (denature, 15 sec), 45-55C (anneal, 30 sec), and 20C (ligate). Do about 5-10 cycles, add bit more ligase, and ATP, do 5-10 more cycles .
- Clean up.
- Rescue PCR with end primers, using ligation cycling product as template.
Notes
This works well for relatively for short fragments (300-500 bp). For longer sequences, use PCR Overlap Extension of the fragments.
References
Rouillard et al. Gene2Olig: oligonucleotide design for in vitro gene syntehsis. [1]
