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.
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== Procedure ==
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== LCR Procedure ==
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[[Image:Primer_Design.JPG|300px|right]]
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# 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.
# 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.
# Dilute the oligos to 100uM.
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== Notes ==
== Notes ==
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This works well for relatively for short fragments (300-500 bp). For longer sequences, use [[PCR Overlap Extension]] of the fragments.
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[[Image:Primer_Design.JPG|300px|right]]
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* 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. 
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* This works well for relatively for short fragments (300-500 bp). For longer sequences, use [[PCR Overlap Extension]] of the fragments.
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== References ==
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==
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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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References ==
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Ro
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uillard 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]
[[Category:Protocol]]
[[Category:Protocol]]
[[Category:DNA]]
[[Category:DNA]]
[[Category:In vitro]]
[[Category:In vitro]]

Revision as of 13:14, 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.

LCR Procedure

  1. 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.
  2. Dilute the oligos to 100uM.
  3. Kinase the oligos.
  4. Ligation cycle:
  • For Thermostable Taq ligase, cycle between:
    1. 95C (denature),
    2. 45-55C (anneal),
    3. 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):
    1. 95C (denature, 15 sec),
    2. 45-55C (anneal, 30 sec),
    3. 20C (ligate).
  • Clean up.
  • Rescue PCR with end primers, using ligation cycling product as template.
  • Gel extract.
  • T-vector or TOPO clone.

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.
  • This works well for relatively for short fragments (300-500 bp). For longer sequences, use PCR Overlap Extension of the fragments.

==

References ==

Ro uillard et al. Gene2Olig: oligonucleotide design for in vitro gene syntehsis. [1]

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