Corum:DNA Hybridization: Difference between revisions
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Sean P Corum (talk | contribs) m (New page: '''~~~~''': ==Overview== Hybridization of ssDNA oligonucleotides. ==Materials== * 5 μL 2mM sense oligonucleotide * 5 μL 2mM antisense oligonucleotide ==Procedure== # Combine sense an...) |
Sean P Corum (talk | contribs) m (→Procedure) |
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# Cool on bench 5 min. | # Cool on bench 5 min. | ||
# 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. | # 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 H<sub>2</sub>O, 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 H<sub>2</sub>O = 200 μL 10μM linker | |||
#* 20 μL 10μM linker + 180 μL H<sub>2</sub>O = 200 μL 1μM linker | |||
#* 20 μL 1μM linker + 180 μL H<sub>2</sub>O = 200 μL 100nM linker | |||
# Store at -40 °C in latest "Primer" box. | |||
==Notes== | ==Notes== | ||
Revision as of 15:15, 25 June 2012
SC 20:55, 19 June 2012 (EDT):
Overview
Hybridization of ssDNA oligonucleotides.
Materials
- 5 μL 2mM sense oligonucleotide
- 5 μL 2mM antisense oligonucleotide
Procedure
- 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.
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
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References
Relevant papers, books, and websites
Contact
or instead, discuss this protocol.
