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Karmella's BioBrick Cloning
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02/23/13
- Type IIS Assembly, PCR: LOV-H2B+his
Type IIS Assembly, PCR: LOV-H2B+his
- H2B = 378 bp, ggH2B f/r (Template: H2B-GFP from Addgene); note: reverse primer adds a 6-his tag (18 bp)
- LOV = 429 bp, ggLOV f/r (Template: LOV from Addgene)
- pSB1A3 = ~2000, gg0001/0002 v2
PCR
- 95°C, 3 min.
- [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
- 72°C, 3 min.
- 4°C, ∞
Reagent
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Volume
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Expected: 1. H2B = 396 2. LOV = 429 3, 4. pSB1A3 = ~2000
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10 μL/lane; 1% agarose; Ladder
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DNA(plasmid) |
0.2 μL
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primer 1 |
1.0
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primer 2 |
1.0
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2x GoTaq |
25.0
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dH2O |
22.8
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50.0 μL
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Purification
- Clean with Zymo DNA c&c; elute w/ 20 μL dH2O
- Measure [DNA]
Sample
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OD 260
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260/280
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ng/μL
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1. H2B+his |
### |
### |
###
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2. LOV |
### |
### |
###
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3. pSB1A3 |
### |
### |
###
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4. pSB1A3 |
### |
### |
###
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Dilutions: to final concentration of 20 fmol/μL
DNA
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length (bp)
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ng/μL
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Vol.
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+ dH2O
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pSB1A3 |
2000 |
77.4 |
6.7 |
13.3
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H2B+his |
186 |
17.0 |
2.8 |
17.2
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LOV |
2520 |
66.9 |
9.8 |
10.2
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Golden Gate Reactions
- hPCD + BL01 + pSB1A3
- pSB1A3
Reagent |
1 |
2
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gg2 pSB1A3 |
1.0 |
1.0
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gg3 hPCD |
1.0 |
---
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gg4 BL01 |
1.0 |
---
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10x ligase buffer |
1.0 |
1.0
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NEB T4 lgase |
0.25 |
0.25
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BsmBI |
0.5 |
0.5
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dH2O |
5.25 |
7.25
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10.0 |
10.0
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Thermal cycling
- [45°C, 2 min.; 16°C, 5 min.] x25
- 60°C, 20 min.
- 80°C, 20 min.
- 4°C, ∞
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