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

Note: The restriction mixes below are calculated for 10 µl final volume for the digest -- 2 µl restriction mix + 8 µl DNA. We do not add any further water to the reaction but instead use a standard (rather low) concentration of DNA. Especially in case of restriction B, it is critical that the DNA sample has been eluted in water, and not in Elution buffer (TRIS) which would otherwise interfere with the restriction buffer.

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

Thanks to the high Glycerol content, the restriction mix can be stored at -20 for, at least, a couple of weeks.

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

Thanks to the high Glycerol content, the restriction mix can be stored at -20 for, at least, a couple of weeks.

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

Thanks to the high Glycerol content, the restriction mix can be stored at -20 for, at least, a couple of weeks.

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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