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Team:Hawaii/Initial Synth. Oligo Assembly
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==Results== | ==Results== | ||
| - | ===First attempt===[[Image:First assembly.jpg |left|thumb|200px|First assembly attempt. Ten microliters loaded into each well of a ~3% agarose gel ran at 95V for 1 hr.]] | + | ===First attempt=== |
| + | [[Image:First assembly.jpg |left|thumb|200px|First assembly attempt. Ten microliters loaded into each well of a ~3% agarose gel ran at 95V for 1 hr.]] | ||
Nice bands observed for annealed oligos. Bands appear to be the correct size (<100bp) but we can't tell if anneal worked (EtBr doesn't tell us if these are ssDNA or dsDNA bands). Smear is probably from loading way too much DNA (see discussion). Possible faint band for pnir ligation. No bands for slr2016 and pilA ligation, probably because way too much DNA was added for the ligation reaction. | Nice bands observed for annealed oligos. Bands appear to be the correct size (<100bp) but we can't tell if anneal worked (EtBr doesn't tell us if these are ssDNA or dsDNA bands). Smear is probably from loading way too much DNA (see discussion). Possible faint band for pnir ligation. No bands for slr2016 and pilA ligation, probably because way too much DNA was added for the ligation reaction. | ||
==Discussion== | ==Discussion== | ||
Revision as of 04:00, 5 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 1 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
- Reference: Quick Ligation Kit from NEB.
Results
First attempt
Nice bands observed for annealed oligos. Bands appear to be the correct size (<100bp) but we can't tell if anneal worked (EtBr doesn't tell us if these are ssDNA or dsDNA bands). Smear is probably from loading way too much DNA (see discussion). Possible faint band for pnir ligation. No bands for slr2016 and pilA ligation, probably because way too much DNA was added for the ligation reaction.
