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December 5, 2012
Results from last week's ligation
- None of the colonies on any plates grew
- Will retry cloning for each (H2B, LOV) by using the Gibson assembly method
- Repeat PCR of H2B and LOV inserts with new primers
- Note: Primers add a XbaI site upstream and the SpeI site downstream
- Final Gibson assembly products
- pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
- pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end
- H2B-GFP plasmid + 1A3/H2B fwd + H2B/LOV rev
- LOV plasmid + H2B/LOV fwd + LOV+his/1A3 rev
- H2B-GFP plasmid + 1A3/LOV fwd + LOV/H2B rev
- LOV plasmid + LOV/H2B fwd + H2B+his/1A3 rev
Performed table reactions twice: once for those used in H2B-LOV construct, once for those used in LOV-H2B construct
| Reagents |
H2B |
LOV
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| Plasmid DNA |
0.5 μL |
0.5
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| primer 1 (10 μM) |
1.0 |
1.0
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| primer 2 (10 μM) |
1.0 |
1.0
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| 2x GoTaq mix |
12.5 |
12.5
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| dH2O |
10.0 |
10.0
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| Total |
25.0 |
25.0
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PCR program:
- 95°C, 3 min.
- [95°C, 30 sec; 57°C, 30 sec.; 72°C, 30 sec.] x35 cycles
- 72°C, 3 min.
- 4°C ∞
- Clean up PCR products using the Zymo Clean and Concentrator kit
- Be sure to elute using 15 μL dH2O
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