IGEM:Paris Bettencourt 2012/Protocols/oligonucleotide inserts

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Oligonucleotide Inserts

Design considerations. Make sure that:

either primer will not form a stable internal hairpin structure, ΔG <-3 kcal/mole

either primer does not contain a EcoRI, PstI, SpeI, XbaI, NotI, or BstEII site outside of the flanking regions

Order 100 nmol DNA oligo with 5' phosphorylated ends and PAGE purification from IDT

Phosphorylation of the 5' ends

With the Fermentas PNK Kit

Note: Skip this step if you ordered phosphorylated oligos

Mix:

  • 2 μL 10 μM sense oligo
  • 2 μL 10 μM anti-sense oligo
  • 2 μL 10xPNK (polynucleotide kinase buffer A)
  • 2 μL 10mM ATP
  • 1 μL T4 polynucleotide kinase (PNK)
  • 10 μL distilled water

to give 20 μL total volume

Incubate at 37°C for 30 mins

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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