Corum:DNA Hybridization

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==Overview==
==Overview==
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Hybridization of ssDNA oligonucleotides to create dsDNA linker.
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Hybridization of complimentary ssDNA oligonucleotides to create dsDNA linker.
==Materials==
==Materials==
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* 5 μL 2mM antisense oligonucleotide
* 5 μL 2mM antisense oligonucleotide
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NOTE: If X = nmol of lyophilized, add X/2 μL water to obtain 2mM final concentration.
+
NOTE: If X = nmol of lyophilized oligonucleotide, add X/2 μL water to obtain 2mM final concentration.
==Procedure==
==Procedure==
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# Combine sense and antisense oligonucleotides. Vortex and spin.
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# Combine 5 μL sense oligonucleotide and 5 μL antisense oligonucleotide. Vortex and spin.
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# Double seal tube airtight with paraffin and press firmly in weighted holder.
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# Double seal tube airtight with parafilm and press firmly in weighted holder.
# Boil 800 mL water in a 1L beaker.
# Boil 800 mL water in a 1L beaker.
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# Cool on bench 3 min to ~95 °C.
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# 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.

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

  1. Combine 5 μL sense oligonucleotide and 5 μL antisense oligonucleotide. Vortex and spin.
  2. Double seal tube airtight with parafilm and press firmly in weighted holder.
  3. Boil 800 mL water in a 1L beaker.
  4. Wait 1 min.
  5. Place weighted holder with sealed oligonucleotides into the beaker.
  6. 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.
  7. 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
  8. 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

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