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Team:Hawaii/Initial Synth. Oligo Assembly
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(New page: ==Protocol== ===Hybridization of Parts=== #Mix: #:3 μl 100 µM sense oligo #:3 μl 100 µM anti-sense oligo #:3 μl 10 x PNK (polynucleotide kinase) buffer #:2 μl 10mM ATP #:2 μl T...)
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(New page: ==Protocol== ===Hybridization of Parts=== #Mix: #:3 μl 100 µM sense oligo #:3 μl 100 µM anti-sense oligo #:3 μl 10 x PNK (polynucleotide kinase) buffer #:2 μl 10mM ATP #:2 μl T...)
Newer edit →
Revision as of 03:01, 4 July 2008
Contents |
Protocol
Hybridization of Parts
- Mix:
- 3 μl 100 µM sense oligo
- 3 μl 100 µM anti-sense oligo
- 3 μl 10 x PNK (polynucleotide kinase) buffer
- 2 μl 10mM ATP
- 2 μl T4 polynucleotide kinase (PNK)
- 17 μl distilled water
- Total volume = 30 μl
- Incubate at 37C for 1.5 hours.
- Add 4 μl 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 (approx. 30 minutes)
- Reference: Pam Silver Lab. [http://openwetware.org/wiki/Silver:_Oligonucleotide_Inserts| Oligonucleotide Inserts.]
DNA Ligation
- Combine 4 ul of construct one with 4 ul of construct 2.
- Add 2 ul of nanopure H20.
- Add 10 ul of 2x quick ligation reaction Buffer. In order to avoid shearing the DNA, mix by resuspending very slowly.
- Add 1 ul of Quick T4 DNA ligase and mix thoroughly by resuspending very slowly.
- Incubate at room temperature for 5 minutes.
- Cool on ice then transform, or store at -20oC
