pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end
Dephosphorylation (11/23/12)
> Dephosphorylation (Roche)
pSB1A3 - X/S = 2000 bp
Reagent
Volume
DNA (clean digest)
11.0 (~200 ng)
10x phos. buffer
1.5
phosphatase
0.5
dH2O
2.0
15 μL
Incubate at 37°C/ 10 min.
Heat inactivate at 75°C/ 2 min.
[final] = 200 ng/μL / 15 μL = ~13 ng/μL
Ligation
Use 20 ng of vector for each ligation = 1.5 μL pSB1A3
Use 1x moles of H2B insert, relative to vector: 1xμL H2B insert = 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 60 ng/μL H2B = at least 0.07 μL H2B
Use 1x moles of LOV insert, relative to vector: 1xμL LOV insert = 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 62 ng/μL LOV = at least 0.07 μL LOV
Since the calulated amounts are so small and a volume of 5 μL is needed to add to the Gibson assembly mix, each value was multiplied by 2 and rounded for simplicity
H2B-LOV
LOV-H2B
Negative Control
H2B-LOV H2B insert DNA (1x mol vector)
1.0 μL
0
0
H2B-LOV LOV insert DNA (1x mol vector)
1.0 μL
0
0
LOV-H2B H2B insert DNA (1x mol vector)
0 μL
1.0
0
LOV-H2B LOV insert DNA (1x mol vector)
0 μL
1.0
0
Vector DNA (39 ng)
3.0
3.0
3.0
Gibson master mix
15.0
15.0
15.0
Total volume
20.0
20.0
18.0
Mix the reaction(s) thoroughly by flicking the tube. Incubate at 50°C for 1 hour.