DNA Synthesis from Oligos

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(PCR Synthesis Procedure)
(PCR Synthesis Procedure)
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:# 95°C (denature, 20 sec)  
:# 95°C (denature, 20 sec)  
:# 72°C (anneal and extend, 40 sec)
:# 72°C (anneal and extend, 40 sec)
-
::*Because you are using the entire oligo as a primer, the annealing temperature is unusually high , so annealing and extention can be done in the same step.<br>  
+
::*Because you are using the entire oligo as a primer, the Tm will be unusually high. Because of this, annealing and extention can be done in the same step.<br>  
6. Clean up PCR product to remove enzyme and primers.<br>
6. Clean up PCR product to remove enzyme and primers.<br>
7. Gel extract.<br>
7. Gel extract.<br>

Revision as of 13:03, 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 (see the notes on primer design below).
2. Dilute the oligos to 100uM.
3. Kinase the oligos.
4. Thermo-cycle:

  • For Thermostable Taq ligase: do as many cycles as necessary (10-30).
  1. 95C (denature)
  2. 45-55C (anneal)
  3. 65C (ligate)
  • 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

1. Design oligos for your gene, of both strands (see the notes on primer design below).
2. Dilute the oligos to 100uM.
4. Thermo-cycle:

  1. 95°C (denature, 20 sec)
  2. 45-55°C (anneal, 20 sec)
  3. 72°C (extend, 20 sec)

5. Take 2μL of this Syntehsis product and do a rescue PCR with the 5' end primers.

  1. 95°C (denature, 20 sec)
  2. 72°C (anneal and extend, 40 sec)
  • Because you are using the entire oligo as a primer, the Tm will be unusually high. Because of this, annealing and extention can be done in the same step.

6. Clean up PCR product to remove enzyme and primers.
7. Gel extract.
8. 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.
  • 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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