SC 20:55, 19 June 2012 (EDT):
Hybridization of ssDNA oligonucleotides.
- 5 μL 2mM sense oligonucleotide
- 5 μL 2mM antisense oligonucleotide
- Combine sense and antisense oligonucleotides. Vortex and spin.
- Seal tube airtight with paraffin and place in weighted holder.
- Boil 400 mL water in a 500 mL beaker.
- Cool on bench 5 min.
- Place weighted holder with sealed oligonucleotides into the beaker.
- 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.
- 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.
- 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
- Store at -40 °C in latest "Primer" box.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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