DNA Synthesis from Oligos

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(LCR Synthesis Procedure)
(LCR Synthesis Procedure)
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== LCR Synthesis Procedure ==
== LCR Synthesis Procedure ==
1. Design oligos for your gene, of both strands.<br>  
1. Design oligos for your gene, of both strands.<br>  
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2. Dilute the oligos to 100uM.
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2. Dilute the oligos to 100uM.<br>
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# Kinase the oligos.
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3. Kinase the oligos.<br>
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# Ligation cycle:
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4. Ligation cycle:<br>
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* For Thermostable Taq ligase, cycle between:  
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(:)* For Thermostable Taq ligase: do as many cycles as necessary (10-30).
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##95C (denature),  
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# 95C (denature),  
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##45-55C (anneal),  
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# 45-55C (anneal),  
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##65C (ligate). Do many as necessary (10-30).
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# 65C (ligate). Do many as necessary (10-30).
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* For T4 ligase, cycle between(Do about 5-10 cycles, add bit more ligase, and ATP, do 5-10 more cycles):
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(:)* For T4 ligase: do 5-10 cycles, add more ligase and ATP, and do 5-10 more.
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##95C (denature, 15 sec),  
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# 95C (denature, 15 sec),  
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##45-55C (anneal, 30 sec),  
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# 45-55C (anneal, 30 sec),  
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##20C (ligate).  
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# 20C (ligate).  
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# Clean up.
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5. Clean up PCR product to remove enzyme and primers.
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# Rescue PCR with end primers, using ligation cycling product as template.
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6. Rescue PCR with end primers, using ligation cycling product as template.
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# Gel extract.
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7. Gel extract.
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# T-vector or TOPO clone.
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8. Clone
== PCR Synthesis Procedure ==
== PCR Synthesis Procedure ==

Revision as of 12:30, 15 June 2009

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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

1. Design oligos for your gene, of both strands.
2. Dilute the oligos to 100uM.
3. Kinase the oligos.
4. Ligation cycle:
(:)* For Thermostable Taq ligase: do as many cycles as necessary (10-30).

  1. 95C (denature),
  2. 45-55C (anneal),
  3. 65C (ligate). Do many as necessary (10-30).

(:)* For T4 ligase: do 5-10 cycles, add more ligase and ATP, and do 5-10 more.

  1. 95C (denature, 15 sec),
  2. 45-55C (anneal, 30 sec),
  3. 20C (ligate).

5. Clean up PCR product to remove enzyme and primers. 6. Rescue PCR with end primers, using ligation cycling product as template. 7. Gel extract. 8. 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]

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