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Corum:DNA Hybridization: Difference between revisions
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Sean P Corum (talk | contribs) m (→Procedure) |
Sean P Corum (talk | contribs) m (→Procedure) |
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==Procedure== | ==Procedure== | ||
# Combine sense and antisense oligonucleotides. Vortex and spin. | # Combine sense and antisense oligonucleotides. Vortex and spin. | ||
# | # Double seal tube airtight with paraffin and press firmly in weighted holder. | ||
# Boil | # Boil 800 mL water in a 1L beaker. | ||
# Cool on bench | # Cool on bench 3 min to ~95 °C. | ||
# Place weighted holder with sealed oligonucleotides into the beaker. | # 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. | # 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 | # Perform two 1/100 dilutions (label all tubes clearly): | ||
#* 2 μL 1mM linker + 198 μL H<sub>2</sub>O = 200 μL 10μM linker | #* 2 μL 1mM linker + 198 μL H<sub>2</sub>O = 200 μL 10μM linker | ||
#* 2 μL 10μM linker + 198 μL H<sub>2</sub>O = 200 μL 100nM linker | #* 2 μL 10μM linker + 198 μL H<sub>2</sub>O = 200 μL 100nM linker | ||
