Team:Freiburg/Protocols/Gibson Assembly

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Gibson Assembly

Protocol for the assembly of PCR products with overhangs (adapted from AG Weber protocol)

material: Gibson Master Mix Aliquots
time: 90 min

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1. prepare 5µl of DNA-Mix (calculate voulumes using the equations below or use the prepared worksheet: Stefan's ExcelSheet
2. add DNA Mix to Gibson Master Mix
3. incubate on RT for 5 min
4. use 5-7µl for transformation of competent E.coli cells

Equations

V (Bb) = 12 ng/kb * l (Bb) / c (Bb)
V (Ins) = 12 ng/kb * l (Ins) * X / c (Ins)

X = Ratio Insert to Backbone
z.B. X = 4, if Ins : Bb = 4 : 1

Gibson master mix

A) ISO buffer

amount	ingredient	remarks
1.5 g	PEG-8000
3 ml	Tris-HCl (1 M, pH 7.5)	dissolve 12.1 g Tris in 100 ml dH2O, adjust pH to 7.5 with conc. HCl
300 µl	DTT (1 M)	dissolve 1.54 g DTT in 10 ml dH2O
150 µl	MgCl2 (2 M)	dissolve 4.06 g MgCl2 in 10 ml dH2O
300 µl	NADNa (100 mM)	dissolve 0.02 g NADNa in 300 µl dH2O
4 x 60 µl	dNTPs (100 mM, each)
up to 6 ml	dH2O

aliquot á 350 µl

B) Assembly master mix

Work on ice!

volume	ingredient
690 µl	dH2O
320 µl	ISO buffer
160 µl	Taq ligase (NEB, 40 U/µl)
20 µl	Q5 Polymerase (NEB, 2 U/µl)
10 µl	T5 Exonuclease (NEB, 0.64 U/µl)*

* dilute 3.2 µl T5 Exonuclease (10 U/µl) in 46.8 µl 1x T5-buffer

aliquot á 15 µl

protocols old