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.
• Complete digestion is critical for a low background -- at least initially, check your digests on a gel!
• The digest volume can be reduced further to 5 µl (4 µl DNA + 1 µl restriction mix). This leaves no sample for a gel though.
• The whole protocol can be performed in a 96-well PCR plate

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

Perform assemblies on 96-well plate

1. multidispense restriction mix A into row A
2. multidispense restriction mix B into row B
3. add 8 µl parts DNA [50ng/µl] to rows A and B
   • Example: you want to couple part 1 to part 2 and, in parallel, part x to part y. Put part 1 into A1, part 2 into B1, part x into A2, part y into B2
4. program & run restriction + heat shock on a PCR device
5. use multi-channel pipette to copy 4 µl from A and B into row D
6. add 2 µl pre-digested vector backbone into each well of D
7. multi-dispense 10µl ligation mix into D
8. seal plate and run ligation protocol on PCR device
9. put plate on ice
10. pipette (gently!) 25 µl competent cells into each well of row F
11. copy 2 µl from ligation row D into cell row F
12. run first half of transformation (20'@4C; 30"@42C; 10'@4C) on PCR device
13. dilute 25 µl cells into 200 µl S.O.C. medium in 2 ml Eppendorf tubes and continue classic transformation protocol

Notes

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References

Example reference

1. ISBN:0879697164

Contact

• Raik

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