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 Karmella's BioBrick Cloning
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12/11/12
- Note: colonies from retransformation of pSB1A3-RFP look red
- 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
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30 μL/lane, 1% agarose; Ladder
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| DNA (plasmid) |
15.0 μL
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| 10x buffer |
3.0
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| enzyme 1 |
1.0
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| enzyme 2 |
1.0
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| dH2O |
10.0
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| |
30 μL --> 37°C/ ~30 min.
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- Measure conc.'s of DNA purified from gel (Zymo kit)
| Sample |
OD260 |
260/280 |
ng/μL
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| 1. KAH11 (E/P) |
0.019 |
1.96 |
18.7
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| 2. C-His (E/P) |
0.047 |
1.82 |
46.8
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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)
- Note: pellets are red
- Digest 10 μL of each with X/S for Gibson cloning (Brady and Vi)
- Note: combined mini preps afterwards; concentration = 60 ng/μL
| Reagent
|
Volume
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Expected:
3. pSB1A3 = 2155
4. pSB1A3 = 2155
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15 μL/lane; 1% agarose; Ladder
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| DNA(plasmid) |
10.0 μL
|
| 10X buffer |
3.0
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| 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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- Gel purification: combined the two slices
- Concentration: 260 = 0.015, 260/280 = 2.2, ng/μL = 15.1
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Karmella's BioBrick Cloning
