DNA Hybridization-PDF

Overview

Hybridization of complimentary ssDNA oligonucleotides to create dsDNA linker.

Materials

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

NOTE: If X = nmol of lyophilized oligonucleotide, add X/2 μL water to obtain 2mM final concentration.

Procedure

1. Combine 5 μL sense oligonucleotide and 5 μL antisense oligonucleotide. Vortex and spin.
2. Double seal the tube airtight with parafilm and press firmly in a weighted holder.
3. Boil 800 mL water in a 1L beaker.
4. Wait 1 min.
5. Place the weighted holder with sealed oligonucleotides into the beaker.
6. Incubate on a bench overnight or until water is about room temperature (~4 hr). At the end of this process, the complimentary ssDNAs are hybridized into a single dsDNA linker with sticky ends at 1mM final concentration.
7. Perform two 1/100 dilutions (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.