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 Karmella's BioBrick Cloning
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12/11/12
- Assembly: KAH111 + C-His (trouble shooting for Behzad, start from plasmids)
- Minipreps: Rene's pSB1A3-RFP (2); cut with X/S for Brady and Vi's Gibson cloning
Assemblies
- KAH111/C-His: 5' part/(E/P)/size + C-His/(E/P)/3600
| Reagent
|
Volume
|
30 μL/lane, 1% agarose; Ladder
|
| DNA (plasmid) |
15.0 μL
|
| 10x buffer |
3.0
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| enzyme 1 |
1.0
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| enzyme 2 |
1.0
|
| dH2O |
10.0
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| |
30 μL --> 37°C/ ~30 min.
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| Sample |
OD260 |
260/280 |
ng/μL
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| 1. KAH11 (E/P) |
--- |
--- |
---
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| 2. C-His (E/P) |
--- |
--- |
---
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| Ligation |
Plate results (lig : neg crtl) 12/12/12
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| 1. KAH111(E/P)/size, 16 ng + C-His(E/P)/3600, 25ng |
new BioBrick #:1 (Pick #)
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| 2. C-His(E/P)/ 25 ng |
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| |
1 |
2 |
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| Insert DNA |
1.0 |
--- |
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| Vector DNA |
0.5 |
0.5 |
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| 2x lgn buf (Roche) |
5.0 |
5.0 |
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| T4 ligase (NEB) |
1.0 |
1.0 |
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| dH2O |
2.5 |
3.5 |
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10 μL |
10 μL |
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Minipreps
- pSB1A3-RFP (retransformation from 12/10/12)
- Concentrations: (1) 260 = , 260/280 = , ng/μL = ; (2) 260 = , 260/280 = , ng/μL = ;
- Digest 10 μL of each with X/S for Gibson cloning (Brady and Vi)
| Reagent
|
Volume
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Expected:
3. pSB1A3 = size
4. BB2 = size
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15 μL/lane; 1% agarose; Ladder
|
| DNA(plasmid) |
10.0 μL
|
| 10X buffer |
3.0
|
| XbaI |
1.0
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| SpeI |
1.0
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| dH2O |
15.0
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| |
15 μL --> 37°C/ ~30 min.
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Karmella's BioBrick Cloning
