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| | | dH<sub>2</sub>O || 2.0 | | | dH<sub>2</sub>O || 2.0 |
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| - | | || 15 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~13 ng/μL | + | | || 15 μL |
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| - | * Heat inactivate .... | + | * Incubate at 37°C/ 10 min. |
| | + | * Heat inactivate at 75°C/ 2 min. |
| | + | * [final] = 200 ng/μLuL / 15 μL = ~13 ng/μL |
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Revision as of 17:17, 21 November 2012
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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
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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 ∞
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
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| DNA | 8.0 | 8.0
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| XbaI | 1.0 | 1.0
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| SpeI | 1.0 | 1.0
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| 10x buffer | 3.0 | 3.0
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| dH2O | 10.0 | 10.0
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| Total vol. | 30. 0 | 30.0
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- Run entire 30 μL on a 1% gel (use the big fat-tooth well comb)
- Cut band out of gel (use UV box)
Results from gel recovery:
| Sample | 260 | 260/280 | ng/μL
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| H2B - X/S | ### | ### | ###
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| LOV - X/S | ### | ### | ###
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- H2B = 410
- LOV = 461
Dephosphorylation
> Dephosphorylation (Roche)
- pSB1A3 - X/S =
| Reagent | Volume
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| DNA (clean digest) | up to 11.0 (~200 ng)
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| 10x buffer d.p. | 1.5
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| phosphatase | 0.5
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| dH2O | 2.0
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| | 15 μL
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- Incubate at 37°C/ 10 min.
- Heat inactivate at 75°C/ 2 min.
- [final] = 200 ng/μLuL / 15 μL = ~13 ng/μL
Ligation
- Use 20 ng of vector for each ligation = 1.5 μL pSB1A3
- Use 2x moles of H2B insert, relative to vector: xμL H2B insert = 3 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / ### ng/μL H2B = 1.28 μL H2B
- Use 2x moles of LOV insert, relative to vector: xμL LOV insert = 3 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / ### ng/μL LOV = 0.8 μL LOV
| | H2B | LOV | Negative Control
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| Insert DNA (2x mol vector) | ### μL | ### | ---
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| Vector DNA (50 ng) | 1.5 | 1.5 | 1.5
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| 2x Roche Rapid Ligation buffer | 5.0 | 5.0 | 5.0
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| New England Biolabs T4 ligase | 1.0 | 1.0 | 1.0
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| dH2O | ### | ### | ###
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| TOTAL | 10.0 | 10.0 | 10.0
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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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