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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
|
| dH2O |
10.0 |
10.0
|
| Total |
25.0 |
25.0
|
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 products, 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
Assemblies
- H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp
- Digests:
- In a 30 μL reaction, cut H2B with XbaI and SpeI
- In a 30 μL reaction, cut LOV with XbaI and SpeI
| Reagent |
H2B PCR |
LOV PCR
|
| DNA |
8.0 |
8.0
|
| XbaI |
1.0 |
1.0
|
| SpeI |
1.0 |
1.0
|
| 10x buffer |
3.0 |
3.0
|
| dH2O |
10.0 |
10.0
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| Total vol. |
30. 0 |
30.0
|
- Run entire 30 μL on a 1% gel (use the big fat-tooth well comb)
- Cut band out of gel (use UV box)
- H2B = 410
- LOV = 461
Dephosphorylation
Ligation
- Use 20 ng of vector for each ligation = 1 μL pSB1A3
- Use 2x moles of H2B insert, relative to vector: xμL H2B insert = 2 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 6.384 ng/μL H2B = 1.28 μL H2B
- Use 2x moles of LOV insert, relative to vector: xμL LOV insert = 2 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 11.492 ng/μL LOV = 0.8 μL LOV
| |
H2B |
LOV |
Negative Control
|
| Insert DNA (2x mol vector) |
1.0 μL |
1.0 |
0
|
| Vector DNA (50 ng) |
1.0 |
1.0 |
1.0
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| 2x Roche Rapid Ligation buffer |
7.0 |
7.0 |
7.0
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| New England Biolabs T4 ligase |
1.0 |
1.0 |
1.0
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| dH2O |
4.0 |
4.0 |
5.0
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| TOTAL |
14.0 |
14.0 |
14.0
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Mix the reaction(s) thoroughly by flicking the tube.
Incubate at room temperature for 10 minutes.
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Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
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