NanoBio: Oligonucleotide Inserts

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(Reworded the ordering thingy, added link to oligocalc)
m (reminder to mix oligos to 100 µM)
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== Hybridize ssDNA to form ds insert (start here if you ordered phosphorylated oligos(right move!))==
== Hybridize ssDNA to form ds insert (start here if you ordered phosphorylated oligos(right move!))==
 +
*Make oligos 100 µM
*Mix:
*Mix:
**3 uL 100 µM sense oligo
**3 uL 100 µM sense oligo

Revision as of 15:38, 10 June 2008

Design of oligonucleotide inserts

  • The sense oligo should be of the form:
    5' CTAGA(coding sequence)ACTAGTAGCGGCCGCTGCA 3'
    The bolded sequences will be the overhanging "sticky" ends.
  • The anti-sense oligo should be of the form:
    5' GCGGCCGCTACTAGT(reverse complement of coding sequence)T 3'
  • Design considerations. Make sure that:
    • either primer will not form a stable internal hairpin structure, ΔG < -3 kcal/mole. You can use OligoCalc
    • either primer does not contain a EcoRI, PstI, SpeI, XbaI, NotI, or BstEII site outside of the flanking regions
    • the insert for the forward primer does not begin with TC (or else a DAM I site (GATC) is formed, the A is methylated and XbaI cannot cut at its site)
  • Order 25 nmol DNA oligo with 5' phosphorylated ends (click 5' Mods at the bottom) from IDT.

Note: if your oligo insert is large 40+ bp, use PAGE purification (it's a little slower and more expensive).

Hybridize ssDNA to form ds insert (start here if you ordered phosphorylated oligos(right move!))

  • Make oligos 100 µM
  • Mix:
    • 3 uL 100 µM sense oligo
    • 3 uL 100 µM anti-sense oligo
    • 3 uL 10 x PNK (polynucleotide kinase) buffer (in common buffer stocks)
    • 3 uL 0.5 M NaCl
    • 18 uL distilled water

to give 30 µL total volume

  • Place in boiling water bath for 2 min., then remove water bath from the heat source and allow the DNA (still in the water bath) to cool to room temperature.
  • 1x PNK buffer (NEB catalog # B0201S) contains: 70 mM Tris-HCl, 10 mM MgCl2, 5 mM Dithiothreitol, pH 7.6.

Phosphorylation of 5' ends & hybridization (only if you didn't order 5'phosphorylated oligos)

  • Note: it is really just better in terms of time & efficiency to order 5' phosphorylated oligos. Really.
  • Mix:
    • 3 uL 100 µM sense oligo
    • 3 uL 100 µM anti-sense oligo
    • 3 uL 10 x PNK (polynucleotide kinase) buffer
    • 2 uL 10mM ATP
    • 2 uL T4 polynucleotide kinase (PNK)
    • 17 uL distilled water

to give 30 uL total volume

  • Incubate at 37C for 1.5 hours.
  • Add 4 uL 0.5 M NaCl.
  • Place in boiling water bath for 2 min., then remove water bath from the heat source and allow the reaction (still in the water bath) to cool to room temperature.
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