DNA Synthesis from Oligos
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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 thermo-cycling.
This synthesis can be accomplished using two methods: Ligation Chain Reaction (LCR) or Polymerase Chain Reaction (PCR). While both protocols are similar, they have some distinct differences which will be described here.
- 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.
- Gel extract.
- T-vector or TOPO clone.
This works well for relatively for short fragments (300-500 bp). For longer sequences, use PCR Overlap Extension of the fragments.
Rouillard et al. Gene2Olig: oligonucleotide design for in vitro gene syntehsis.