Editing Join Fragments by Taq Ligase (section)

== <br/>TPA ==

'''Design of TPA oligonucleotides'''<br/>For each DNA fragment to be assembled, four oligonucleotides were ordered: long forward (LF), short forward (SF), long reverse (LR) and short reverse (SR). The long primers (LF and LR) were analogous to that used in GA, and they defined the overlap region, whereas the short primers stopped exactly before the overlap. The TPA oligonucleotides design algorithm was as follows:

The melting temperature (TM) of the overlap region (Figure (Figure1B)1B) was set at 50°C as predicted by IDT's OligoAnalyzer using the following modified conditions: Oligo concentration = 0.015 μM, Na+ = 50 mM, Mg2+ = 10 mM. All junctions used in an assembly should have the same TM.

<br/>As much as possible, G/C locks (i.e. the nucleotide closest to the nick being a G or C) were used at both sides of the nicks when choosing junction sequences. In situations where such locks were not possible, consecutive T/A close to the nicks should be avoided.

<br/>The overlap regions of the long primers were defined by their TM rather than by length. Long primers with TM = 50°C junctions were usually GA compatible.<br/>The short primers (SF or SR) were ordinary PCR primers adjacent to the overlap sequences.

<br/>'''Two-step assembly: preparation of fragments'''<br/>For each DNA fragment to be assembled, two PCR products were generated: one with primers LF and SR, the other with primers SF and LR. PCR reactions were performed using either KOD Xtreme Hotstart (Merck, Darmstadt, Germany) or Q5 (NEB, Ipswich, MA, USA) DNA polymerase according to the respective manufacturer's recommended protocol. Depending on PCR purity, either gel purification (QIAquick Gel Extraction Kit, Qiagen, Hilden, Germany) or PCR purification (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany) were used to clean up the products. The two PCR products for each fragment were mixed in 1:1 molar ratio, to a final concentration of 40 fmol/μl DNA (40 fmol/μl ≈ 24 ng/kb/μl DNA) in 1× CutSmart buffer (NEB, Ipswich, MA, USA). A lower final concentration (down to 10 fmol/μl) was used for some fragments when the higher final concentration could not be achieved due to low PCR yield. An excel worksheet was used to facilitate the calculations (see Supplementary Protocol for details). If plasmid template was used in the PCR, 0.5 μl DpnI was added to eliminate the plasmid template so as to reduce the transformation background. The re-annealing step that generates overhangs was carried out using the following temperature profile: 37°C for 30 min (for DpnI digest), 98°C for 2 min, 85°C for 2 min (at a ramping rate of 0.1°C/s), 75°C for 2 min (0.1°C/s), 65°C for 2 min (0.1°C/s), 55°C for 2 min (0.1°C/s) and 8°C hold (0.1°C /s). The annealed DNA fragments were stored at 4°C until the assembly step.

'''Two-step assembly: assembly of fragments'''<br/>The assembly was carried out by hybridizing the re-annealed fragments. All the re-annealed DNA fragments were mixed in 1:1 molar ratio, to a final concentration of 4 fmol/μl/fragment (4 fmol/μl ≈ 2.4 ng/kb/μl) in 1× CutSmart buffer. The hybridization step that generates nicked circular plasmid was carried out using the following temperature profile: 65°C (TM + 15) for 10 s, 55.5°C (TM + 5.5) for 1–2 h and 8°C hold (0.1°C/s). The hybridized nicked plasmid was stored at 4°C until E. coli transformation.

'''One-step assembly'''<br/>In one-step assembly, all PCR products for the construct were put together in one-pot at a final concentration of 2 fmol/μl/product in 1× CutSmart buffer, 0.5 μl DpnI was added if necessary. The one-step assembly was carried out using the following temperature profile: 37°C for 30 min (DpnI digest), 98°C for 2 min, 85°C for 2 min (0.1°C/s), 75°C for 2 min (0.1°C /s), 65°C for 2 min (0.1°C/s), 55.5°C (TM + 5.5) for 1–2 h (0.1°C/s) and 8°C hold (0.1°C/s). The hybridized nicked plasmid was stored at 4°C until E. coli transformation.

'''E. coli transformation'''

Use E coli competent cells by chemical method for transformation