13-Fragment Golden Gate Assembly Protocol using SapI (NEB #R0569)


Overview

Golden Gate Assembly of 13 fragments using SapI restriction enzyme with T4 DNA Ligase and subsequent transformation into NEB 10-beta Competent E. coli (High Efficiency). 

Materials

  • T4 DNA Ligase (NEB #M0202)
  • SapI (NEB #R0569)
  • Destination Plasmid (used provided)
  • NEB 10-beta Competent E. coli (NEB #C3019)
  • NEB 10-beta/Stable Outgrowth Medium (NEB #B9035)
  • Selective LB Agar plates

Reaction Set-up

Set up 20 µl assembly reaction as follows:

REAGENT ASSEMBLY REACTION
Destination Plasmid(1)  3 nM (final concentration)
Amplicon Inserts(2) 3 nM(3) each amplicon (final concentration)
SapI (NEB #R0569), 10 U/µl 1.5 μl (15 units)
T4 DNA Ligase (NEB #M0202), 2000 U/µl 0.25 μl (500 units)
T4 DNA Ligase Buffer (NEB #B0202(10X) 2 μl 
Nuclease-free H2O (NEB #B1500) to 20 μl(4)

(1) Destination vector must possess SapI restriction sites at both ends of the insert sequence and in the proper orientation.
(2) Amplicon inserts must possess 5 ́ flanking bases (6 recommended) and SapI restriction sites at both ends of the amplicon and
in the proper orientation.
(3) The NEBiocalculator® Tool (nebiocalculator.neb.com) can be used for molarity calculations.
(4) Can be increased to 25 μl volume if required due to DNA component volumes; add additional 0.5 μl T4 DNA Ligase Buffer
(10X).

Reaction Assembly Protocol

SUGGESTED ASSEMBLY PROTOCOL 
(37°C, 5 min → 16°C, 5 min) x 30 → 60°C, 5 min → 4°C(5)

(5) Cool reaction to 4°C prior to transformation, or store completed assembly reactions at -20°C.

Transformation

  1. For each assembly, thaw a 50 µl tube of NEB 10-beta competent E. coli cells on ice for 5–10 min.
  2. Add 2 µl of the assembly reaction; gently mix by flicking the tube 4-5 times.
  3. Incubate on ice for 30 min.
  4. Heat shock at 42°C for 30 sec.
  5. Place back on ice for 5 min.
  6. Add 950 µl of room temperature NEB 10-beta/Stable Outgrowth Medium (NEB #B9035). Incubate at 37°C for 60 min., shaking vigorously (250 rpm) or using a rotation device.

Plating

  1. Warm selective LB agar plates at 37°C for 15 min.
  2. Mix the cells thoroughly by flicking the tube and inverting, then spread 100 µl outgrowth(6) onto each plate.
  3. Incubate the plates overnight at 37°C, or 24 hrs at 30°C, or 48 hrs at 25°C.
(6) Outgrowth dilution may be required to obtain well spaced colony forming units

 

Figure 1: High capacity Golden Gate assembly with T4 DNA ligase and SapI.


(A) Schematic of the 13-fragment lac operon cassette test system. (B) Results of the assembly reactions. Four replicate experiments were carried out to quantify the number of colony-forming units harboring correct and incorrect assembly products per μL of E. coli outgrowth plated (0.002 μL of the assembly reaction). On average, 91% of the observed transformants harbored correctly assembled products. (C) Representative agar plate with blue and white colonies. Blue transformants harbor correct assembly constructs, and white transformants harbor inaccurate assembly products. Pryor, J. M. et al. (2020). PLoS Onehttps://doi.org/10.1371/journal.pone.0238592

 

Figure 2: Golden Gate Assembly fidelity predictions as a function of the overhang pairs in the assembly reaction.


The GetSet tool was used to estimate the fidelity of assembly reactions containing up to 30 overhang pairs with T4 DNA ligase and SapI. Overhang pairs were selected using Data-optimized Assembly Design (DAD; blue).  Pryor, J. M. et al. (2020). PLoS Onehttps://doi.org/10.1371/journal.pone.0238592


References