Silver: Oligonucleotide Inserts
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Design of oligonucleotide inserts
- The sense oligo should be of the form:
5' CTAGA(coding sequence)ACTAGTAGCGGCCGCTGCA 3' - 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
- 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 100 nmol DNA oligo with 5' phosphorylated ends and PAGE purification from IDT.
Phosphorylation of 5' ends & hybridization (only if you didn't order 5'phosphorylated oligos)
- 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.
Hybridize ssDNA to form ds insert
- Mix:
- 3 uL 100 µM sense oligo
- 3 uL 100 µM anti-sense oligo
- 3 uL 10 x PNK (polynucleotide kinase) buffer
- 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.