SC 20:55, 19 June 2012 (EDT):
Hybridization of ssDNA oligonucleotides.
- 5 μL 2mM sense oligonucleotide
- 5 μL 2mM antisense oligonucleotide
NOTE: If X = nmol of lyophilized, add X/2 μL water to obtain 2mM final concentration.
- Combine sense and antisense oligonucleotides. Vortex and spin.
- Double seal tube airtight with paraffin and press firmly in weighted holder.
- Boil 800 mL water in a 1L beaker.
- Cool on bench 3 min to ~95 °C.
- 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 as single dsDNA linker with sticky ends at 1mM final concentration.
- 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
- 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.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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