Gibson Assembly is rather novel method for assembling DNA fragments with overlapping overhangs. The operating mode of this method is devided into three major parts:

1. An exonuclease removes bases from the 5' end of each DNA strand.
2. Complementary regions of different DNA strands can anneal and a polymerase fills up the gaps.
3. The fragments are ligated together.

To enable these three steps, DNA strands with compatible ends of about 32 bp are needed. Those can either be incorporated by primer overhangs or by gene synthesis.
A 5 µl mix of the DNA parts that are supposed to be assembled is prepared. Its composition is calculated based on the length and concentration of every single fragment. The insert(s) should at least be contained in a 4 - 8 fold molar amount of the antibiotic resistance containing backbone.

Assembly of DNA fragments with overlapping regions (adapted from AG Weber protocol)

Material: Gibson Master Mix Aliquots
Time: 90 min

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1. Prepare 5 µl of DNA-Mix (calculate voulumes using the equations below or use the prepared worksheet.
2. Add the DNA Mix to Gibson Master Mix.
3. Incubate for 5 min at RT.
4. Use 5 - 7 µl for transformation of competent E.coli cells.

Backbone:
V (Bb) = 12 ng/kb * l (Bb) / c (Bb)

Insert(s):
V (Ins) = 12 ng/kb * l (Ins) * X / c (Ins)

X = Ratio Insert to Backbone
for example: X = 4, if Ins : Bb = 4 : 1

B) Gibson 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

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