Ligation and enzyme-independent assembly methods

		Number of fragments (fidelity)	Enzyme required	PCR Primer Design	Ref
Ligase independent
	Gibson Assembly			Requirements
	CPEC	5 fragments for a 9 kb plasmid (90%)	DNA polymerase
	In-fusion, Clontech	5 fragments for a 5 kb plasmid (90%)	In-fusion HD enzyme mix
	Hot fusion	8 fragments for a 8 kb plasmid (90%)	T5 Exonuclease, DNA polymerase	17–30 bp homology	Hot Fusion 2
	MOE-PCR	7 fragments for a 7 kb plasmid (25%)	DNA polymerase
	SLIC	10 fragments for a 8 kb plasmid (20%)	T4 DNA polymerase, RecA
	SLiCE	7 fragments for a 5 kb plasmid (90%)	Escherichia coli extract
	USER  uracilspecific excision reagent cloning	8 fragments for a 11 kb plasmid (60%)	USER enzyme mix
	CAME		T4 DNA polymerase, or Pfu DNA polymerase, or λ exonuclease then T4 DNA polymerase
Enzyme-independent assembly methods
	EFC	2 fragments for a 3 kb plasmid (90%)
	PIPE	2 fragments for a 5 kb plasmid (90%)
	SLIC	2 fragments for a 15 kb plasmid
	TPA	10 fragments for a 7 kb plasmid (80%)		4 pairs of primers

Hot Fusion Protocol

 5X pre-assembly buffer

1 ml of 5x pre-assembly buffer (0.5 M Tris pH 7.5, 50 mM MgCl₂, 1 mM each dNTP, 50 mM DTT, 25% PEG-8000) 

0.5 ml of 1 M Tris pH 7.5

50 µl of 1 M MgCl₂

100 µl of 10 mM dNTP mix (NEB)

50 µl of 1 M DTT

250 mg PEG-8000

150 µl of dH2O.

Store at -20˚C.

2X Hot Fusion buffer 

160 µl of 5X pre-assembly buffer

0.3 µl of 10 u/µl T5 exonuclease (Epicentre, Madison, WI)

10 µl of 2 u/µl Phusion polymerase

230 µl of dH2O.

Final: 400 µl  

The final concentration

0.0075 u/µl T5 exonuclease

 0.05 u/µl Phusion Hot Start DNA polymerase.

Aliquots:  10 µl aliquots,  store  at -20˚C with no more than 10 times freezer thaw cycles.

Hot Fusion reaction

1 µl of a linearized vector (30–50 ng/µl) 

2–3 µl of PCR insert (30–100 ng/ µl)

1 µ ml of 2 x Hot Fusion Buffer.

H₂O to 20 µl

For multiple fragment assembly (3–4 fragments), 1 µl of each insert (20–50 ng/µl) should be used.

Incubate  in a thermocycler for 1 hour at 50˚C, then slowly ramp  down to 20˚C in 5 minutes (0.1˚C per second), and hold at 4˚C .

Transformation: 

Electroporation: use 1 µl.

Notes:

One fragment cloning, incubate at 37˚C for 5 minutes and 5 minutes at 68˚C is enough for generate colonies for screening.

However, for assembly of multiple fragments the best results were still observed using the regular conditions 

for single enzyme digested vector, NEB Gibson Assembly yields over 10 fold more background colonies than Hot Fusion due to self ligation

Multipe fragments assembly,  incubate 1 hour at 50˚C.  the cloning efficiency of Gibson assembly drops to 70% due to more background colonies produced by self-ligation ,  whereas Hot Fusion reached 92% efficiency.

Optimization of DNA to vector ratio or adjusting DNA concentration is not necessary in most cases, particularly for single or two fragment cloning (with a range of 20 ng to 100 ng). However the efficiency could be enhanced with a vector to insert ratio of 3:1. An excess of inserts (i.e., 10 fold), particularly for more than three fragment assembly does not help for the efficiency, instead yields fewer colonies probably due to competition of each DNA molecule for enzymes. This could cause imperfect chew-backs for each molecule at both 59 and 39 ends (due to enzyme limitations).. Seven fragments (more than 6 kb) has been successfully assembled by Hot Fusion in one step, indicating that Hot Fusion is an efficient method to build complex constructs or entire vectors.  

1. Digest vector with restriction enzyme(s) overnight, and purify the linearized vector with a commercial PCR purification kit. Elute the DNA with elution buffer or 10 mM TrisCl, pH 8.0-8.5. Do not elute the DNA with water or TE. Measure the concentration of the vector.

2. Amplify your gene of interest by PCR using primers with ≥ 15 mer homology extension to the linearized vector end. We usually use 15 bp homology for single fragment cloning, and 20 bp homology for multiple fragment cloning. Purify the linearized vector with a commercial PCR purification kit. Elute the DNA with elution buffer or 10 mM TrisCl, pH 8.0-8.5. Do not elute the DNA with water or TE. Measure the concentration of the insert(s).

3. Mix the linearized vector and insert at a molar ratio of 1:2 in a 1.5 ml tube. An example is shown as follows. (Vector to insert molar ratio of 1:1 to 1:7 works well, but we usually use 1:2 for single fragment cloning, 1:2:2 for multiple fragments cloning. An example of 3 fragments cloning is shown below with vector: insert 1: insert 2 molar ratio is 1:2:2 as shown in Fig. 4A)

4. Add 0.2 µl of T4 DNA polymerase (3 U/µl, NEB) to the mixture and incubate at  room temperature for 2.5 min. We found that 0.5 µl (1.5 U) of T4 DNA polymerase gives the best result, but 0.2 µl gives more than sufficient number of colonies.

5. Put the reaction mixture on ice immediately to stop the reaction and incubate on ice for 10 min.

6. Thaw chemically competent E. coli cells on ice for ~10 min.

7. For single fragment cloning, gently mix the cells with 1-2 µl of the reaction mixture and incubate the cells on ice for 20 min. For multiple fragments cloning, mix the cells with 3-5 µl of the reactant.

8. Incubate the cells on ice for 20 min.

9. Heat shock the cells at 42 oC for 45 sec.

10. Incubate the cells on ice for 2 min.

11. Add 900 (or 950) µl of LB broth to 100 (or 50) µl of the cells and transfer the cells to 15 ml round-bottom tube.

12. Incubate the cells at 37oC for 1 hr.

13. Plate the cells on agar plates containing suitable antibiotics (eg, 100 µg/ml ampicillin). We usually spread 10 to 20 µl of the cells onto an agar plate to get optimal number of colonies per plate for single fragment cloning, and 100 µl per plate for multiple fragments cloning. In case of 10-20 µl spreading, cells needs to be further diluted with 80~90 µl LB broth for suitable spreading.

14. Incubate the plates at 37oC for 16 hr and analyze the colonies.

PIPE cloning without purification

1. Amplify the insert and vector by PCR using a minimum amount of vector plasmid template (0.5 ng template/50 µL reaction) according to manufacturer’s instructions with a high fidelity DNA polymerase, such as Phusion Hot Start II High Fidelity  DNA Polymerase, but omit the final extension step. An enhancer such as 5% DMSO can be added to increase robustness.
An example thermal cycling program for Phusion:
98 °C 3 min,
(98 °C 30 s 63 °C 30 s 72 °C 45 s/kb) x 35

2. Check the products on an agarose gel for successful amplification. If vector plasmid amplification failed, cut the vector with a restriction enzyme within the multiple-cloning site and repeat the PCR using more template. If non-specific products are observed, you may wish to optimise the PCR conditions or gel extract the correct fragment for maximum cloning efficiency. For gel extraction of the correct fragment, avoid exposure to damaging short-wave UV light, such as by using methylene blue staining with visible light, or loading duplicate samples, cutting the gel in half between them and marking the transilluminated half as a reference.

3. Combine 5 µL of vector and 5 µL of insert PCR product (or vary accordingly for varying yields by band intensity to get close to a 2:1 molar ratio) with 10 U (0.5 µL) of DpnI and incubate at 37 °C for 1-3 hr. To save time, digestion can be performed during gel electrophoresis.

4. Transform ultracompetent bacteria with the digest mixture up to 10% of the final volume of cells according to manufacturer’s instructions. For example, add 2 µL DNA to 18 µL of XL10 Gold cells 

PIPE cloning with purification and SLIC using T4 DNA polymerase

1. Perform PCR as above.

2. Analyse products by agarose gel electrophoresis as above.

3. Purify products using a single PCR purification column with a minimal elution (~30 µL) volume. Alternatively, purify separately, quantify by spectroscopy and combine 0.025 pmol vector (~50-100 ng) and 0.0625 pmol insert (~50-200 ng) per 10 µL.

4. Make up with 1x CutSmart Buffer or equivalent and digest with 10 U (0.5 µL) of DpnI/10 µL reaction volume for 1-3 hr at 37 °C.

5. Separate into three fractions and incubate 10 µL samples for 0 (PIPE), 5 or 10 min at 25 °C with 0.75 U (0.25 µL) T4 DNA polymerase, then place on ice for 10 min.

6. Transform bacteria as above.

Overlap extension cloning

1. Perform PCR as above.

2. Analyse products by agarose gel electrophoresis as above.
If non-specific products or primer-dimers are present it is essential to remove them by PCR optimisation, gel extraction as above, or T4 treatment.

3. Column purify megaprimer and quantify by spectroscopy. 
For products <500 bp, 1 µL of PCR product can be used directly for thermal cycling without purification. Reduced efficiencies may be observed for larger fragments.

4. Perform overlap extension linear amplification.
The optimal megaprimer concentration to use is: <350 bp, ~100-300 fmol (5-15 nM); 
<1.5 kb ~50-100 fmol (2.5-5 nM); >1.5 kb, ~25-50 fmol (1.25-2.5 nM).
To remove primer-dimers with T4 DNA polymerase, treat a 10 µL fraction of 1x SmartCut buffer containing ten-fold the optimum megaprimer in 10 µL, incubate
with 3 U (1 µL) of T4, incubate for 30 min at 25 °C and immediately use 5 µL for overlap extension.

Perform thermal cycling according to manufacturer’s instructions with a high fidelity, non-strand-displacing DNA polymerase, such as Phusion Hot Start II High Fidelity
DNA Polymerase, a 20 µL reaction volume, 25 ng of plasmid template and 5% DMSO.

An example thermal cycling program for Phusion:
72 °C 5 min (to blunt the megaprimer),
98 °C 3 min,
(98 °C 30 s 63 °C 30 s 72 °C 45 s/kb) x 30

5. Dilute 5 uL of product with 5 uL CutSmart Buffer (to maximise DpnI activity and reduce inhibition by PCR buffer) and 20 U (1 µL) DpnI and incubated for 1-3 hr at
37 °C.

6. Transform bacteria as above.

SLIC sub-cloning using T4 DNA polymerase treated inserts with RecA

https://www.nature.com/protocolexchange/protocols/170#/related-articles

1. Digest 2 μg of vector with restriction enzymes. Gel purify the vector and isolate the DNA using QIAEX II gel extraction kit. Quantitate the vector.

2. Amplify inserts using Taq DNA polymerase. Set up a 100 μl PCR reaction with 250 μM of each dNTP, 0.5 μM of each primer, and 2.5 U of Taq DNA polymerase (from Eppendorf). Cycle as follows: 94°C for 45 seconds; 30 cycles of 94°C for 45 seconds, 54°C for 45 seconds, and 72°C for 1 minute; 72°C for 10 minutes. Add 20 U of DpnI to 100 μl of PCR products after PCR, incubate at 37°C for 1 hour (not necessary if going from a MAGIC vector to ColE1 origin). Purify the PCR products by QIAquick PCR purification column. Quantitate the inserts. We typically have 20 bp homology between the vector and the inserts.

3. Take 1 μg of the vector and 1 μg of the inserts and treat separately with 0.5 U of T4 DNA polymerase in T4 buffer (NEB) plus BSA in a 20 μl reaction at room temperature for 30 minutes. Stop the reaction by adding 1/10 volume of 10 mM dCTP and leave on ice.

4. Set up a 10 μl annealing reaction using 1:1 insert to vector ratio with 3 ng or less of a 3.1 kb vector (0.0015 pmol), 1x ligation buffer (NEB), appropriate amount of insert, 20 ng of RecA protein (Epicentre Biotechnologies) and water. Incubate at 37°C for 30 minutes. Leave on ice or store in -20°C.

5. Add 5 μl of the annealed mixture into 150 μl of BW23474 chemical competent cells, incubate on ice for 30 minutes, heat shock at 42°C for 45 seconds, return to ice for 2 minutes, add 0.9 ml of SOC and recover at 37°C for 1 hour.

6. Plate 100 μl onto plates containing the appropriate antibiotics and incubate at 37°C overnight. (Cl-Phe is used for vectors that have PheS-G294 between restriction enzyme sites. We have found that most background comes from uncut vector in our preps and therefore we can select against it with Cl-Phe. However, Cl-Phe is not an essential step and usually only has a 2-fold effect on background.)

Complementary annealing mediated by exonuclease (CAME)

Use 30~50 ng of the vector and 30~50 ng of insert for CAME cloning. CAME reactions were typically carried out in a total volume 10 UL with 1 unit of enzyme and its corresponding buffer (Supplemental Table S1).

Purify PCR products (to eliminate dNTPs when Pfu or T4 DNA polymerase is used to recess the DNA ends).

(1) for T4 DNA polymerase, recess the DNA using T4 DNA polymerase at room temperature for 3 min, heat inactivate the T4 DNA polymerase at 75 oC for 5 min and anneal the DNA at 50 oC for 20~30 min; (2) for λ exonuclease, recess the DNA using λ exonuclease at 37 oC for 5 min, heat inactivate the λ exonuclease at 75 oC for 5 min and anneal the DNA at 50 oC for 20~30 min;

(3) for Pfu DNA polymerase, recess and anneal the DNA using Pfu DNA polymerase for 30 min at 50 oC; or (4) in cases of cloning of an insert whose ends are flanked by undesired sequences, recess the DNA using λ exonuclease at 37 oC for 30 min, heat inactivate the λ exonuclease at 75 oC for 5 min and anneal the DNA at 50 oC for 20~30 min, followed by using T4 DNA polymerase at 37 oC for 15 min to recess the 30 overhangs and fill-in gaps.

T4 DNA polymerase exhibits the highest exonuclease activity and that it can digest almost the entire 450 bp DNA fragment in 5 min.   λ exonuclease and Pfu DNA polymerase show intermediate and the lowest activity. CAME using T4 DNA polymerase generates up to four times more white colonies than using λ exonuclease and Pfu DNA polymerase. 

Pful Buffer: : 20 mM Tris (pH 8.8), 10 mM KCl, 10 mM NH4)2SO4, 2 mM MgSO4, 0.1% TritonX-100

 T4 DNA polymerase Buffer: 67 mM Tris (pH 8.8), 17 mM (NH4)2SO4, 6.7 mM MgCl2, 1 mM DTT.

λ Exonuclease Buffer: 67 mM Glycine-KOH (pH 9.5), 2.5 mM MgCl2, 50 μg/ml BSA, 0.2 mM dNTPs. λ exonuclease prefers 5’ phosphorylated substrates, T4 polynucleotide kinase (T4 PNK) ishould be included to phosphorylate PCR products.

a single exonuclease such as T4 DNA polymerase and λ exonuclease is ineffective for cloning the target fragment when a mixture of many other DNA fragments are present. Hoever, sequential treatment with λ exonuclease and T4 DNA polymerase generated the highest number of colonies with correct insert.  Complementary sequences longer than 20 bp are required for this cloning