Editing Join Fragments by Taq Ligase (section)

== [[File:TPA.png]] ==
Number of fragments (fidelity)Enzyme requiredReference


{|
|-
| colspan="4" | Ligase-independent assembly methods
|-
| CPEC
| 5 fragments for a 9 kb plasmid (90%)
| DNA polymerase
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B17 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B17]
|-
| In-fusion
| 5 fragments for a 5 kb plasmid (90%)
| In-fusion HD enzyme mix
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B18 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B18]
|-
| Hot fusion
| 8 fragments for a 8 kb plasmid (90%)
| T5 Exonuclease, DNA polymerase
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B19 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B19]
|-
| MOE-PCR
| 7 fragments for a 7 kb plasmid (25%)
| DNA polymerase
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B22 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B22]
|-
| SLICa
| 10 fragments for a 8 kb plasmid (20%)
| T4 DNA polymerase, RecA
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B20 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B20]
|-
| SLiCEa
| 7 fragments for a 5 kb plasmid (90%)
| Escherichia coli&nbsp;extract
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B21 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B21]
|-
| USER
| 8 fragments for a 11 kb plasmid (60%)
| USER enzyme mix
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B10 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B10]
|-
|-
| colspan="4" | Enzyme-independent assembly methods
|-
| EFC
| 2 fragments for a 3 kb plasmid (90%)
| 
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B23 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B23]
|-
| PIPE
| 2 fragments for a 5 kb plasmid (90%)
| 
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B13 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B13]
|-
| SLICb
| 2 fragments for a 15 kb plasmid
| 
| [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B20 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B20]
|-
| TPAa
| 10 fragments for a 7 kb plasmid (80%)
| 
| https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499748/#B13
|}

The largest demonstrated number of fragments is shown for each method with the corresponding fidelity in bracket.

aThe largest demonstrated assembly sizes are as indicated except for SLIC (12 kb from 2 fragments), SLiCE (24 kb from 2 fragments) and TPA (31 kb from 5 fragments).

bSLIC has an enzyme independent protocol.







'''PIPE cloning'''<br/>0.025 pmol of vector and 0.0625 pmol insert purified products (2.5∶1 insert∶vector ratio) were digested for 3 hr at 37°C with 10 U of DpnI in 10 µL CutSmart Buffer (NEBuffer 4.1/0.1 mg/mL BSA), diluted 1∶1 with 1× HF buffer (to control for buffer composition between purified and unpurified products, as 1× HF buffer halved transformation efficiency), and 2 µL used to transform 18 µL of XL10 Gold ultracompetent cells (Agilent Technologies) according to manufacturer's instructions.

'''SLIC'''<br/>DpnI digested purified PIPE products were incubated at 25°C for 5 min with 0.75 U of T4 DNA polymerase, immediately placed on ice for 10 min, diluted 1∶1 with ice-cold 1× HF Buffer, and 2 µL used for transformation as described above.

'''OEC'''<br/>20 µL OEC reactions were performed using 10–250 fmol of insert product −84 bp: 250 fmol (12.5 nM); 350 bp: 100 fmol (5 nM); 1.4 kb: 25 fmol (1.25 nM); 4.3 kb: 10 fmol (0.5 nM) - as megaprimer and 25 ng of pUC18/Kan vector template with 5% DMSO, 0.4 U of Phusion Hot Start II High Fidelity DNA polymerase, and cycling conditions: 72°C 5 min (to blunt the megaprimer), 98°C 3 min, (98°C 30 s 63°C 30 s 72°C 3 min) ×30. 5 µL fractions were diluted 1∶1 with CutSmart Buffer (to control for buffer and assist DpnI activity), digested for 3 hr at 37°C with 20 U of DpnI, and 2 µL used for transformation as described above.
[[Category:Antibodies]] [[Category:Protocols]]