Prbbbb:fusion assembly v1

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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
  • pSB1AC3F (CRG) -- BBa_J18901
  • pSB1AK3F (CRG) -- BBa_J18902
  • pSB1AT3F (CRG) -- BBa_J18903
• 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

1. mix 8 µl part A [50 ng/µl] with 2 µl restriction A
2. mix 8 µl part B [50 ng/µl] with 2 µl restriction B
3. mix 8 µl vector [25 ng/µl] with 2 µl restriction C (or use pre-digested stock)
4. incubate for 2h @ 37°C
5. 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

1. mix 4 µl part A digest + 4 µl part B digest + 2 µl vector digest
2. add (as last component!) 10 µl ligation mix (2x)
3. incubate 1h @ 16°C; 10' @ 65deg;C
4. use 2 µl for transformation

Notes

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References

Example reference

1. ISBN:0879697164

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