Prbbbb:fusion assembly v1
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Overview
Materials
- enzymes: AgeI, EcoRI, PstI, NgoMIV (=NgoMI), T4_DNA_Ligase
- NEB_buffers: NEBuffer EcoRI, NEBuffer 1, NEBuffer 4, T4 DNA Ligase buffer
- NEB BSA 100x concentrated
- ddH2O
- linear vector backbone DNA from Prbbbb:vector_pcr; concentration: 25 ng/µl
- DNA Biobrick(s) A (first part), Biobrick(s) B (second part); concentration 50 ng/µl
Procedure
Restriction
restriction mix A (5x concentrated)
1 µl, single reaction | 20 µl | 50 µl | 100 µl | |
---|---|---|---|---|
H2O | 0.25µl | 5 | 12.5 | 25 |
10 x NEBuffer 1 | 0.5µl | 10 | 25 | 50 |
EcoRI 20U/µl | 0.05µl | 1 | 2.5 | 5 |
AgeI 5U/µl | 0.2µl | 4 | 10 | 20 |
restriction mix B (5x concentrated)
1 µl, single reaction | 20 µl | 50 µl | 100 µl | |
---|---|---|---|---|
H2O | 0.2µl | 4 | 10 | 20 |
10 x NEBuffer 4 | 0.5µl | 10 | 25 | 50 |
100 x BSA | 0.05 | 0.1 | 2.5 | 5 |
PstI 10U/µl | 0.125µl | 2.5 | 6.25 | 12.5 |
NgoMI 10U/µl | 0.125µl | 2.5 | 6.25 | 12.5 |
restriction mix C (5x concentrated)
1 µl, single reaction | 20 µl | 50 µl | 100 µl | |
---|---|---|---|---|
H2O | 0.2µl | 4 | 10 | 20 |
10 x NEBuffer EcoRI | 0.5µl | 10 | 25 | 50 |
100 x BSA | 0.05 | 0.1 | 2.5 | 5 |
EcoRI 20U/µl | 0.08µl | 0.16 | 4 | 8 |
PstI 10U/µl | 0.17µl | 3.4 | 8.5 | 17 |
- mix 8 µl part A [50 ng/µl] with 2 µl restriction A
- mix 8 µl part B [50 ng/µl] with 2 µl restriction B
- mix 8 µl vector [25 ng/µl] with 2 µl restriction C (or use pre-digested stock)
- incubate for 2h @ 37°C
- heat inactivate 20' @ 80°C
Ligation
ligation mix (2x concentrated)
10 µl, single reaction | 150 µl | µl | µl | |
---|---|---|---|---|
H2O | 7µl | |||
5 x T4 buffer | 2µl | |||
T4 DNA Ligase | 1 |
- mix 4 µl part A digest + 4 µl part B digest + 2 µl vector digest
- add (as last component!) 10 µl ligation mix (2x)
- incubate 1h @ 16°C; 10' @ 65deg;C
- use 2 µl for transformation
Notes
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References
Example reference
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