NanoBio: Oligonucleotide Inserts: Difference between revisions
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== Design of oligonucleotide inserts== | == Design of oligonucleotide inserts== | ||
*(Optional)Use Gene Designer from DNA 2.0 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 [http://www.dna20.com/tools.php here]. | |||
* | *General idea: Create 2 oligonucleotides that contain your part flanked by the appropriate BioBrick/BioFusion sticky ends. | ||
* | |||
*Design considerations. Make sure that: | *Design considerations. Make sure that: | ||
**either primer will not form a stable internal hairpin structure, ΔG < -3 kcal/mole. You can use [http://www.basic.northwestern.edu/biotools/oligocalc.html OligoCalc] | **either primer will not form a stable internal hairpin structure, ΔG < -3 kcal/mole. You can use [http://www.basic.northwestern.edu/biotools/oligocalc.html OligoCalc] |
Revision as of 12:25, 17 June 2008
Design of oligonucleotide inserts
- (Optional)Use Gene Designer from DNA 2.0 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 (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.