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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
- pSB1A3, gg0001/0002 v2
| Reagent
|
Volume
|
Expected:
1. pSB1A3 = ~2000
2. hPCD = 186
3. BL01 = 2520
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10 μL/lane; 1% agarose; Ladder
|
| DNA(plasmid) |
0.2 μL
|
| primer 1 |
1.0
|
| primer 2 |
1.0
|
| 2x GoTaq |
25.0
|
| dH2O |
22.8
|
| |
50.0 μL
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PCR
- 95°C, 3 min.
- [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
- 72°C, 3 min.
- 4°C, ∞
Purification
- Clean with Zymo DNA c&c; elute w/ 20 μL dH2O
- Measure [DNA]
| Sample
|
OD 260
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260/280
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ng/μL
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| 1. pSB1A3 |
0.126 |
1.888 |
77.357
|
| 2. hPCD |
0.069 |
2.192 |
17.025
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| 3. BL01 |
0.121 |
1.898 |
66.878
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Dilutions: to final concentration of 20 fmol/μL
| DNA
|
length (bp)
|
ng/μL
|
Vol.
|
+ dH2O
|
| pSB1A3 |
2000 |
77.4 |
6.7 |
13.3
|
| hPCD |
186 |
17.0 |
2.8 |
17.2
|
| BL01 |
2520 |
66.9 |
9.8 |
10.2
|
Golden Gate Reactions
- hPCD + BL01 + pSB1A3
- pSB1A3
| Reagent |
1 |
2
|
| gg2 pSB1A3 |
1.0 |
1.0
|
| gg3 hPCD |
1.0 |
---
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| gg4 BL01 |
1.0 |
---
|
| 10x ligase buffer |
1.0 |
1.0
|
| 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
|
| |
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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Karmella's BioBrick Cloning
