Labwork

Protocols

Gibson assembly

1. Principle: One-step isothermal in vitro recombination

Two adjacent DNA fragments sharing terminal sequence 20 bp overlaps were joined into a covalently sealed molecule in a one-step isothermal reaction.

T5 exonuclease removed nucleotides from the 5' ends of doublestranded DNA molecules, complementary single-stranded DNA overhangs annealed.

Phusion DNA polymerase filled the gaps and Taq DNA ligase sealed the nicks.
T5 exonuclease is heat-labile and is inactivated during the 50°C incubation.

Incubation at 50°C during 1 hour --> the product can be used to transform into E. coli directly.

2. Design primer

Primer overlap at least 20 bp with the adjacent fragments (plasmid, fragments)

Order primer: IDT Company
Scale: 25nm DNA oligo
Normalization: None
Purification: standard desalting

The Tm is defined as the temperature in degrees Celsius, at which 50% of all molecules of a given DNA sequence are hybridized into a double strand, and 50% are present as single strands (the melting temperature).
Tm of overlap part must be higher than 50°C.

3. PCR amplification

Using enzyme Phusion High-Fidelity DNA polymerase (the most accurate thermostable polymerase available).

4. Clean PCR, plasmid

• For DNA fragments: Gel purify if it has an unspecific band or purify PCR product by kit. Estimate the concentration.
• For plasmid: after PCR, digest your PCR product by enzyme DpnI (recognize and cut methylated DNA, allow to remove remaining plasmid template, and does not cleave PCR-amplified DNA). Then, purify your PCR plasmid digestion and estimates the concentration.

5. Gibson amplification

• Add the combined fragments (5 µl) to one Isothermal Assembly reaction aliquot (15 µl) and mix by pipetting (20 µl total).
• Not exceeding a total volume of 20 µl. If required, bring to 5 µl with ddH2O. 100 ng of plasmid is recommended.
• Incubate the mix at 50°C for 60 min.
• Transform E. coli DH5α with 10 µl of assembly mix.

6. Transformation in E. coli

Negative control: transform E. coli with the plasmid PCR product digested with DpnI --> know the background.

Isothermal assembly

Isothermal Assembly works by combining a cocktail of exonuclease, polymerase, and ligase to fuse dsDNA fragments with sufficiently (20-120 bp) homologous ends. It leaves no "scar" behind, i.e. you can expect your product to contain the EXACT overlap sequence. The reaction may work with shorter ends (e.g. 15 bp), so long as the annealing temperature is higher than 50C.

To perform isothermal assembly:

1. PCR up your fragments of choice, and purify. Also gel purify the cut vector.
2. Not exceeding a total volume of 5 µl, in a PCR tube, combine fragments at equal molecular ratio. If required, bring to 5 µl with ddH2O. Use approx. 100 ng of plasmid backbone.
3. Add the combined fragments (5 µl) to 1 Isothermal Assembly reaction aliquot (15 µl) and mix by pipetting (20 µl total).
4. Place mix at 50°C for 60 min.
5. Transform with 10 µl of assembly mix.

References:

Gibson et al (2009) Nature Methods 6(5):343-345.

Making Isothermal Assembly Aliquots

5x isothermal assembly reaction buffer (assemble on ice):

From the paper:	Actually added:
3 mL 1M Tris-HCl pH 7.5	3 mL 1M Tris-HCl pH 7.5
150 µL 2M MgCl2	300 µL 1M MgCl2
60 µL 100 mM dGTP	600 µL 10 mM each dNTP
60 µL 100 mM dCTP
60 µL 100 mM dTTP
60 µL 100 mM dATP
300 µL 1M DTT/td>	300 µL 1M DTT
1.5 g PEG-8000	1.5 g PEG-8000
300 µL 100 mM NAD	20 mg NAD
ddH2O to 6 ml	ddH2O to 6 ml

Prepare 320 µL aliquots (18) and freeze all but one. * Label these “5X isotherm buffer”
To the one remaining (320 µL), add:

1.2 µL	T5 Exonuclease
20 µL	Phusion polymerase
160 µL	Taq ligase
700 µL	ddH2O

Prepare 15 µL aliquots (~80) on ice in PCR tubes and store at -20C. These should be good for up to a year.

Shopping List

Buffer

Item	Vendor	Ref
1M Tris-HCl pH 7.5	Sigma	T2319
Magnesium Chloride, 1.00 +/- 0.01M Solution	Affymetrix / USB	78641 10 x 1 ml
Nicotinamide adenine dinucleotide (NAD)	VWR	A1124,0005
DTT, molecular biology grade	FERMENTAS	R0861
Polyethylene Glycol 8000, Powder	USB / Affymetrix	19966
dNTP Mix, 10mM each	Fermentas	R0192 1 ml

Enzymes:

T5 Exonuclease	NEB	M0363S
Taq DNA Ligase	NEB	M0208L
Phusion™ High-Fidelity DNA Polymerase	Thermoscientific	F530-S