IGEM:Paris Bettencourt 2012/Protocols/oligonucleotide inserts: Difference between revisions
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Design considerations. Make sure that: | Design considerations. Make sure that: | ||
either primer will not form a stable internal hairpin structure, ΔG <-3 kcal/mole | 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 | 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 | Order 100 nmol DNA oligo with 5' phosphorylated ends and PAGE purification from IDT | ||
'''Phosphorylation of the 5' ends''' | '''Phosphorylation of the 5' ends''' | ||
With the Fermentas PNK Kit | With the Fermentas PNK Kit | ||
Revision as of 09:11, 30 July 2012
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.
