Hybridization of ssDNA oligonucleotides to create dsDNA linker.
- 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.
- SC 19:27, 24 July 2012 (EDT):
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