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11/21/12
- Repeat PCR of H2B and LOV inserts
- Note: Primers add a XbaI site upstream and the SpeI, NotI, and PstI sites downstream
- H2B-GFP plasmid + BB_H2B fwd + BB_H2B rev
- LOV plasmid + BB_LOV fwd + BB_LOV rev
| Reagents |
H2B |
LOV
|
| Plasmid DNA |
0.5 μL |
0.5
|
| primer 1 (10 μM) |
1.0 |
1.0
|
| primer 2 (10 μM) |
1.0 |
1.0
|
| 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 ∞
Expected:
1. H2B = 410
2. LOV = 461
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Results of the PCR product digests are shown, with H2B on the left and LOV on the right. The gel was loaded with 5 μL samples.
- ---Karmella 15:56, 21 November 2012 (EST): Digest, purification, and ligation
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