DNA Synthesis from Oligos: Difference between revisions
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== Procedure == | == Procedure == | ||
[[Image:Primer_Design.JPG|300px|right]] | [[Image:Primer_Design.JPG|300px|right]] | ||
# 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), | |||
##65C (ligate). Do many as necessary (10-30). | |||
* For T4 ligase, cycle between(Do about 5-10 cycles, add bit more ligase, and ATP, do 5-10 more cycles): | |||
##95C (denature, 15 sec), | |||
##45-55C (anneal, 30 sec), | |||
##20C (ligate). | |||
* Clean up. | * Clean up. | ||
* Rescue PCR with end primers, using ligation cycling product as template. | * Rescue PCR with end primers, using ligation cycling product as template. |
Revision as of 09:39, 15 June 2009
back to protocols | ||
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 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.
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),
- 65C (ligate). Do many as necessary (10-30).
- For T4 ligase, cycle between(Do about 5-10 cycles, add bit more ligase, and ATP, do 5-10 more cycles):
- 95C (denature, 15 sec),
- 45-55C (anneal, 30 sec),
- 20C (ligate).
- Clean up.
- Rescue PCR with end primers, using ligation cycling product as template.
- Gel extract.
- T-vector or TOPO clone.
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]