Corum:DNA Hybridization
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m (New page: '''~~~~''': ==Overview== Hybridization of ssDNA oligonucleotides. ==Materials== * 5 μL 2mM sense oligonucleotide * 5 μL 2mM antisense oligonucleotide ==Procedure== # Combine sense an...) |
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==Overview== | ==Overview== | ||
| - | Hybridization of ssDNA oligonucleotides. | + | Hybridization of complimentary ssDNA oligonucleotides to create dsDNA linker. |
==Materials== | ==Materials== | ||
| - | + | ||
* 5 μL 2mM sense oligonucleotide | * 5 μL 2mM sense oligonucleotide | ||
* 5 μL 2mM antisense oligonucleotide | * 5 μL 2mM antisense oligonucleotide | ||
| + | |||
| + | NOTE: If X = nmol of lyophilized oligonucleotide, add X/2 μL water to obtain 2mM final concentration. | ||
==Procedure== | ==Procedure== | ||
| - | # Combine sense and antisense | + | # Combine 5 μL sense oligonucleotide and 5 μL antisense oligonucleotide. Vortex and spin. |
| - | # | + | # Double seal tube airtight with parafilm and press firmly in weighted holder. |
| - | # Boil | + | # Boil 800 mL water in a 1L beaker. |
| - | # | + | # Wait 1 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 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 H<sub>2</sub>O = 200 μL 10μM 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. | ||
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| + | ==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. | ||
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==Contact== | ==Contact== | ||
Current revision
Contents |
Overview
Hybridization of complimentary ssDNA oligonucleotides to create dsDNA linker.
Materials
- 5 μL 2mM sense oligonucleotide
- 5 μL 2mM antisense oligonucleotide
NOTE: If X = nmol of lyophilized oligonucleotide, add X/2 μL water to obtain 2mM final concentration.
Procedure
- Combine 5 μL sense oligonucleotide and 5 μL antisense oligonucleotide. Vortex and spin.
- Double seal tube airtight with parafilm and press firmly in weighted holder.
- Boil 800 mL water in a 1L beaker.
- Wait 1 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 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.
