Corum:DNA Hybridization

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

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 as single dsDNA linker with sticky ends at 1mM final concentration.
7. Perform two 1/100 dilutions down to 100 nM (label all tubes clearly):
   • 2 μL 1mM linker + 198 μL H₂O = 200 μL 10μM linker
   • 2 μL 10μM linker + 198 μL H₂O = 200 μL 100nM linker
8. Store 1mM, 10μM, and 100nM stocks at -40 °C in latest "Primer" box. Keep an aliquot of the 100nM linker in your own box for use in constructions.

Notes

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References

Relevant papers, books, and websites

1. New England Biolabs

Contact

• Sean P Corum

or instead, discuss this protocol.