Editing Ligation and enzyme-independent assembly methods (section)

== Hot Fusion Protocol ==

[[File:Hot fusion 1.png|RTENOTITLE]][[File:Hot fusion 2.png|RTENOTITLE]]

=== &nbsp;5X pre-assembly buffer ===

1 ml of 5x pre-assembly buffer (0.5 M Tris pH 7.5, 50 mM MgCl<sub>2</sub>, 1 mM each dNTP, 50 mM DTT, 25% PEG-8000)&nbsp;

0.5 ml of 1 M Tris pH 7.5

50 <span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l of 1 M MgCl<sub>2</sub>

100 <span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l of 10 mM dNTP mix (NEB)

50 <span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l of 1 M DTT

250 mg PEG-8000

150 <span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l of dH2O.

Store at -20˚C.

=== 2X Hot Fusion buffer&nbsp; ===

160 <span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l of 5X pre-assembly buffer

0.3 <span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l of 10 u/<span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l T5 exonuclease (Epicentre, Madison, WI)

10 <span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l of 2 u/<span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l Phusion polymerase

230 <span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l of dH2O.

Final: 400&nbsp;<span style="font-size: small; color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif;">µ</span>l&nbsp;&nbsp;

The final concentration

0.0075 u/<span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l T5 exonuclease

&nbsp;0.05 u/<span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l Phusion Hot Start DNA polymerase.

Aliquots:&nbsp; 10 <span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l aliquots,&nbsp;&nbsp;store&nbsp; at -20˚C with no more than 10 times freezer thaw cycles.

=== Hot Fusion reaction ===

1 <span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l of a linearized vector (30–50 ng/<span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l)&nbsp;

2–3 <span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l of PCR insert (30–100 ng/ <span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l)

1&nbsp;<span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span> ml of 2 x Hot Fusion Buffer.

H<sub>2</sub>O to 20&nbsp;<span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µl</span>

For multiple fragment assembly (3–4 fragments), 1 <span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l of each insert (20–50 ng/<span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µ</span>l) should be used.

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

'''Transformation''':&nbsp;

'''Electroporation''':&nbsp;use 1&nbsp;<span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">µl.</span>

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&nbsp;

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

Multipe fragments assembly,&nbsp;&nbsp;incubate 1 hour at 50˚C.&nbsp;&nbsp;the cloning efficiency of Gibson assembly drops to 70% due to more background colonies produced by self-ligation ,&nbsp;&nbsp;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.&nbsp;&nbsp;





[[File:TPA assembly.png|RTENOTITLE]]





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

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

<br/>3. Mix the linearized vector and insert at a molar ratio of 1:2 in a 1.5 ml tube. An&nbsp;example is shown as follows. (Vector to insert molar ratio of 1:1 to 1:7 works well,&nbsp;but we usually use 1:2 for single fragment cloning, 1:2:2 for multiple fragments&nbsp;cloning. An example of 3 fragments cloning is shown below with vector: insert 1:&nbsp;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&nbsp;&nbsp;room temperature for 2.5 min. We found that 0.5 µl (1.5 U) of T4 DNA polymerase&nbsp;gives the best result, but 0.2 µl gives more than sufficient number of colonies.

<br/>5. Put the reaction mixture on ice immediately to stop the reaction and incubate on ice&nbsp;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&nbsp;and incubate the cells on ice for 20 min. For multiple fragments cloning, mix the cells&nbsp;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).&nbsp;We usually spread 10 to 20 µl of the cells onto an agar plate to get optimal number of&nbsp;colonies per plate for single fragment cloning, and 100 µl per plate for multiple&nbsp;fragments cloning. In case of 10-20 µl spreading, cells needs to be further diluted&nbsp;with 80~90 µl LB broth for suitable spreading.

<br/>14. Incubate the plates at 37oC for 16 hr and analyze the colonies.