NanoBio: Oligonucleotide Inserts: Difference between revisions

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== Design of oligonucleotide inserts==
== Design of oligonucleotide inserts==
 
*(Optional)Use Gene Designer from DNA2.0 to create an optimal DNA sequence from a protein coding sequence. This is particularly helpful if you are making a sequence which is repetitive on the amino acid level. You can download this program for free [https://www.dna20.com/tools/genedesigner.php here].
*The sense oligo should be of the form:<br> 5' '''CTAG'''A(coding sequence)ACTAGTAGCGGCCGC'''TGCA''' 3'<br> The bolded sequences will be the overhanging "sticky" ends.
*General idea: Create 2 oligonucleotides that contain your part flanked by the appropriate BioBrick/BioFusion sticky ends.
*The anti-sense oligo should be of the form:<br> 5' GCGGCCGCTACTAGT(reverse complement of coding sequence)T 3'
*Design considerations.  Make sure that:
*Design considerations.  Make sure that:
**either primer will not form a stable internal hairpin structure, &Delta;G < -3 kcal/mole
**either primer will not form a stable internal hairpin structure, &Delta;G < -3 kcal/mole. You can use [http://www.basic.northwestern.edu/biotools/oligocalc.html OligoCalc]
**either primer does not contain a EcoRI, PstI, SpeI, XbaI, NotI, or BstEII site outside of the flanking regions
**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)
**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 100 nmol DNA oligo with 5' phosphorylated ends and PAGE purification from [http://www.idtdna.com IDT].
*Order 25 nmol DNA oligo with 5' phosphorylated ends (click 5' Mods at the bottom) from [http://www.idtdna.com IDT].
*Note: if you're feeling stingy or in a rush and your oligo insert is small ~30 bp, you can skip PAGE purification and the insert will still [[likely]] be okay. For longer inserts, PAGE purification is highly recommended.
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!))==
== Hybridize ssDNA to form ds insert==
*Note: this procedure is appropriate for 5' phosphorylated oligonucleotides only.
*Make oligos 100 µM in distilled water.
*Mix:
*Mix:
**3 uL 100 µM sense oligo
**3 uL 100 µM sense oligo
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*Add 4 uL 0.5 M NaCl.
*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.
*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.
== ==
*Back to [[NanoBio:Protocols|Protocols]].

Latest revision as of 17:30, 5 November 2009

Design of oligonucleotide inserts

  • (Optional)Use Gene Designer from DNA2.0 to create an optimal DNA sequence from a protein coding sequence. This is particularly helpful if you are making a sequence which is repetitive on the amino acid level. You can download this program for free here.
  • General idea: Create 2 oligonucleotides that contain your part flanked by the appropriate BioBrick/BioFusion sticky ends.
  • 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

  • Note: this procedure is appropriate for 5' phosphorylated oligonucleotides only.
  • Make oligos 100 µM in distilled water.
  • 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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