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 Karmella's BioBrick Cloning
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02/23/13
- Type IIS Assembly, PCR: H2B-LOV+his
Type IIS Assembly, PCR: LOV-H2B+his
- H2B = 378 bp, 1A3/H2B fwd + H2B/LOV rev (Template: H2B-GFP from Addgene)
- LOV = 429 bp, LOV fwd + LOV+his/1A3 rev (Template: LOV from Addgene); note: reverse primer adds a 6-his tag (18 bp)
- pSB1A3 = ~2000, gg0001 v2 + gg0002 v2
- Be sure to use the H2B and LOV primers ordered on 2/4/2003. The names are redundant with a previous version.
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
|
Volume
|
Expected:
1. H2B = 378
2. LOV = 447
3, 4. pSB1A3 = ~2000
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10 μL/lane; 1% agarose; Ladder
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| DNA(plasmid) |
0.2 μL
|
| primer 1 |
1.0
|
| primer 2 |
1.0
|
| 2x GoTaq |
25.0
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| dH2O |
22.8
|
| |
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 |
0.04 |
1.667 |
40.4
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| 2. LOV |
0.019 |
1.562 |
18.6
|
| 3. pSB1A3 |
0.069 |
1.754 |
69.4
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| 4. pSB1A3 |
0.085 |
1.817 |
85.5
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Dilutions: to final concentration of 20 fmol/μL (40 μL)
- x = length in bp ÷ measured ng/μL * 0.013 * 40
| DNA
|
length (bp)
|
ng/μL
|
Vol. (x)
|
+ dH2O
|
| pSB1A3 |
2000 |
85.5 |
12.2 |
27.8
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| H2B+his |
396 |
40.4 |
5.1 |
34.9
|
| LOV |
429 |
18.6 |
12.0 |
28.0
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(02/24/13)
Golden Gate Reactions
- LOV + H2B+his + pSB1A3
- pSB1A3
| Reagent |
1 |
2 |
Master mix (x3)
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| gg2 pSB1A3 |
1.0 |
1.0 |
---
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| gg3 LOV |
1.0 |
H2O |
---
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| gg4 H2B+his |
1.0 |
H2O |
---
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| 10x PRO ligase buffer |
1.0 |
1.0 |
3.0
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| NEB T4 lgase |
0.25 |
0.25 |
0.75
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| BsmBI |
0.5 |
0.5 |
1.5
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| dH2O |
5.25 |
5.25 |
15.75
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| |
10.0 |
10.0
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- Aliquot 7.0 mix to each tube; add DNA/ H2O
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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Karmella's BioBrick Cloning
