Corum:DNA Hybridization

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SC 20:55, 19 June 2012 (EDT):

Contents

Overview

Hybridization of ssDNA oligonucleotides.

Materials

  • 5 μL 2mM sense oligonucleotide
  • 5 μL 2mM antisense oligonucleotide

Procedure

  1. Combine sense and antisense oligonucleotides. Vortex and spin.
  2. Seal tube airtight with paraffin and place in weighted holder.
  3. Boil 400 mL water in a 500 mL beaker.
  4. Cool on bench 5 min.
  5. Place weighted holder with sealed oligonucleotides into the beaker.
  6. Incubate on bench overnight or until water is about room temperature (~4 hr). At the end of this process, the complimentary ssDNA's are hybridized into dsDNA linker at 1mM final concentration.
  7. Dilute the 1mM linker 1/10 (~10 μL 1mM linker into ~ 90 μL H2O, may need to be adjusted if some volume is lost and, for example only 9 μL 1mM linker is recovered). Mix and spin.
  8. Continue 1/10 dilutions down to 100 nM (label all tubes clearly):
    • 20 μL 100μM linker + 180 μL H2O = 200 μL 10μM linker
    • 20 μL 10μM linker + 180 μL H2O = 200 μL 1μM linker
    • 20 μL 1μM linker + 180 μL H2O = 200 μL 100nM linker
  9. Store at -40 °C in latest "Primer" box.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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References

Relevant papers, books, and websites

  1. New England Biolabs

Contact

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