Corum:DNA Hybridization: Difference between revisions
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==Overview== | ==Overview== | ||
Hybridization of ssDNA oligonucleotides. | Hybridization of ssDNA oligonucleotides. | ||
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#* 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 | ||
# 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. | # 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. | ||
==Signature== | |||
*'''SC 19:27, 24 July 2012 (EDT)''': | |||
==Notes== | ==Notes== | ||
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Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip. | Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip. | ||
==Contact== | ==Contact== |
Revision as of 16:27, 24 July 2012
Overview
Hybridization of ssDNA oligonucleotides.
Materials
- 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.
Procedure
- 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.
Signature
- SC 19:27, 24 July 2012 (EDT):
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
Contact
or instead, discuss this protocol.