DNA Synthesis from Oligos

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== Notes ==
== Notes ==
This works for relatively well for short fragments (300-500 bp). For longer sequences, use [[PCR Overlap Extension]] of the fragments.
This works for relatively well for short fragments (300-500 bp). For longer sequences, use [[PCR Overlap Extension]] of the fragments.
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== 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 13:33, 29 May 2006

Contents

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 for relatively well 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]

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