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 Karmella's BioBrick Cloning
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01/05/13
- Golden gate: PCR parts, complete protocol for Brady's construct
- Gibson: PCR parts; waiting on BsmBI from NEB
Gibson Assembly
- hPCD + BL01 + V0120
- hPCD + BL01 + pSB1A3
PCR
- V0120, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002)
- pSB1A3, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002)
- hPCD, BBP-hPCD fwd (BL9) / hPCD-BL01 rev (BL7)
- BL01, hPCD-BL01 fwd (BL8) / BL01-BBS rev (BL10)
| Reagent
|
Volume
|
Expected:
G1. V0120 = 3200
G2. pSB1A3 = 2000
G3. hPCD = 186
G4. BL01 = 2520
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10 μL/lane; 1% agarose; Ladder
|
| DNA(plasmid) |
0.5 μL
|
| 10 μM primer 1 |
1.0
|
| 10 μM primer 2 |
1.0
|
| 2x GoTaq mix |
25.0
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| dH2O |
22.5
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| |
50 μL
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- 95°C, 3 min.
- [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30
- 72°C, 3 min.
- 4°C, ∞
Measure DNA Concentrations
| Sample
|
OD 260
|
260/ 280
|
ng/μL
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| G1 V0120 |
0.108 |
1.801 |
108.375
|
| G2 pSB1A3 |
0.101 |
1.849 |
100.553
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| G3 hPCD |
0.034 |
1.876 |
33.586
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| G4 BL01 |
0.113 |
1.828 |
112.79
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Reaction set-up
- Use 0.2 - 0.5 pmol, per NEB's suggestion, [1]
- pmols = ng x 1000 / bp x 650 daltons; ng = (pmols x bp x 650 daltons) / 1000
- Total volume of DNA = 5.0 μL max
1. hPCD + BL01 + V0120
- vector V0120, 0.1 pmol = 208 ng = 1.9 μL (use 1.4 μL)
- 2:1 insert hPCD, 0.2 pmol = 24 ng = 0.7 μL
- 2:1 insert BL01, 0.2 pmol = 328 ng = 2.9 μL
2. hPCD + BL01 + V0120
- vector pSB1A3, 0.1 pmol = 130 ng = 1.3 μL
- 2:1 insert hPCD, 0.2 pmol = 24 ng = 0.7 μL (use 0.8 μL)
- 2:1 insert BL01, 0.2 pmol = 328 ng = 2.9 μL
Gibson reactions:
- hPCD + BL01 + V0120
- V0120
- hPCD + BL01 + pSB1A3
- pSB1A3
| Reagent
|
1 |
2 |
3 |
4
|
| Vector |
1.4 |
1.4 |
1.3 |
1.3
|
| Ins 1 |
0.7 |
--- |
0.8 |
---
|
| Ins 2 |
2.9 |
--- |
2.9 |
---
|
| dH2O |
--- |
3.6 |
--- |
3.7
|
| |
5.0 μL
|
- Add each to one aliquot of 15.0 Gibson mix (Rene); total = 20.0 μL
- Thermal cycler: 50°C,60 min.; 4°C, ∞
Transformation
- (1) Transform: add 2.0 μL to 50 μL DH5α-Turbo; ice/5 min.; plate on warm 100 μg/mL amp. agar
- (2) NEB also suggests diluting 5.0 μL of the Gibson reaction into 15.0 μL dH2O (20.0 total vol.), and using 2.0 μL diluted DNA
- Test both methods (8 plates total)
- Plates 1-4, method 1
- Plates 5-8, method 2
Golden Gate Assembly
- Try new protocol from Dave Savage
- hPCD + BL01 + V0120
- hPCD + BL01 + pSB1A3
- Check the sequences for BsmBI sites!
PCR
- V0120, gg_BBS F (gg0001) / gg_BBP R (gg0002)
- pSB1A3, gg_BBS F (gg0001) / gg_BBP R (gg0002)
- hPCD, gg_BBP-hPCD F (gg0003) / gg_hPCD-BL01 R (gg0004)
- BL01, gg_BL01 F (gg0004) / gg_BL01-BBS R (gg0005)
| Reagent
|
Volume
|
Expected:
gg1. V0120 = 3200
gg2. pSB1A3 = 2000
gg3. hPCD = 186
gg4. BL01 = 2520
|
10 μL/lane; 1% agarose; Ladder
|
| DNA(plasmid) |
0.5 μL
|
| 10 μM primer 1 |
1.0
|
| 10 μM primer 2 |
1.0
|
| 2x GoTaq mix |
25.0
|
| dH2O |
22.5
|
| |
50 μL
|
- 95°C, 3 min.
- [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30
- 72°C, 3 min.
- 4°C, ∞
Measure DNA Concentrations
| Sample
|
OD 260
|
260/ 280
|
ng/μL
|
| gg1 V0120 |
0.099 |
1.834 |
98.549
|
| gg2 pSB1A3 |
0.068 |
1.758 |
67.725
|
| gg3 hPCD |
0.022 |
1.72 |
22.485
|
| gg4 BL01 |
0.056 |
1.707 |
55.883
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Karmella's BioBrick Cloning
