DNA Synthesis from Oligos
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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. | 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. | ||
| - | == LCR Procedure == | + | == LCR Synthesis Procedure == |
| - | # Design oligos for your gene, of both strands | + | # Design oligos for your gene, of both strands. |
# Dilute the oligos to 100uM. | # Dilute the oligos to 100uM. | ||
# Kinase the oligos. | # Kinase the oligos. | ||
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##45-55C (anneal, 30 sec), | ##45-55C (anneal, 30 sec), | ||
##20C (ligate). | ##20C (ligate). | ||
| - | + | # Clean up. | |
| - | + | # Rescue PCR with end primers, using ligation cycling product as template. | |
| - | + | # Gel extract. | |
| - | + | # T-vector or TOPO clone. | |
| + | |||
| + | == PCR Synthesis Procedure == | ||
== Notes == | == Notes == | ||
[[Image:Primer_Design.JPG|300px|right]] | [[Image:Primer_Design.JPG|300px|right]] | ||
| - | * Primer design for the two types of synthesis are different. For a rough determination of primers see the figure to the right. Arrows point in the 5' to 3' direction. | + | * Primer design for the two types of synthesis are different. For a rough determination of primers see the figure to the right. Arrows point in the 5' to 3' direction. |
| + | * Make primers fixed 30-40mers with 15-20mer overlaps; or use the software by Rouillard et al. referenced below. | ||
* This works well for relatively for short fragments (300-500 bp). For longer sequences, use [[PCR Overlap Extension]] of the fragments. | * 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. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15215375] | |
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[[Category:Protocol]] | [[Category:Protocol]] | ||
[[Category:DNA]] | [[Category:DNA]] | ||
[[Category:In vitro]] | [[Category:In vitro]] | ||
Revision as of 13:18, 15 June 2009
| back to protocols | ||
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 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.
LCR Synthesis Procedure
- Design oligos for your gene, of both strands.
- 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.
PCR Synthesis Procedure
Notes
- Primer design for the two types of synthesis are different. For a rough determination of primers see the figure to the right. Arrows point in the 5' to 3' direction.
- Make primers fixed 30-40mers with 15-20mer overlaps; or use the software by Rouillard et al. referenced below.
- 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]
