Corum:DNA Hybridization: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Sean P Corum (talk | contribs) m (→Procedure) |
Sean P Corum (talk | contribs) m (→Overview) |
||
(9 intermediate revisions by the same user not shown) | |||
Line 1: | Line 1: | ||
==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 (~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 | ||
# 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== | ||
Line 28: | Line 31: | ||
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== |
Latest revision as of 16:32, 24 July 2012
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.