24-Fragment Golden Gate Assembly using BsaI-HF®v2 (NEB #R3733)


Overview

Golden Gate Assembly of 24 fragments using BsaI-HFv2 restriction enzyme with T4 DNA Ligase and subsequent transformation into NEB 10-beta Competent E. coli (High Efficiency). 

Materials

  • T4 DNA Ligase (NEB #M0202)
  • BsaI-HFv2 (NEB #R3733)
  • pGGAselect Destination Plasmid*
  • NEB 10-beta Competent E. coli (NEB #C3019)
  • NEB 10-beta/Stable Outgrowth Medium (NEB #B9035)
  • LB Agar plates with chloramphenicol
    * Included in the NEB Golden Gate Assembly Mix (NEB #E1601)

Note: For complex (>10 fragment) assemblies, high efficiencies are achievable with increased ligase and BsaI-HFv2 levels (1000 units T4 DNA Ligase, 30 units BsaI-HFv2), as listed in this protocol. For assemblies involving 10 fragments and less, the standard amounts (500 units T4 DNA Ligase, 15 units BsaI-HFv2) are sufficient. Note the reaction volume of 25 µl is used to allow sufficient volume for precloned insert additions, if needed.

Reaction Set-up

Set up 25 µl assembly reaction as follows:

REAGENTS ASSEMBLY REACTION NEGATIVE CONTROL (IF DESIRED)
pGGAselect Destination Plasmid(1)*, 75 ng/μl 1 μl (75 ng) 1 μl (75 ng)
24 precloned inserts(2) cloned into pMiniT 2.0, 100 ng/ul each plasmid 0.75 µl (75 ng) each, (18 µl total) -
T4 DNA Ligase Buffer (NEB #B0202) (10X) 2.5 μl 2.5 μl
T4 DNA Ligase (NEB #M0202), 2000 U/µl 0.5 μl (1000 units) 0.5 μl (1000 units)
BsaI-HFv2 (NEB #R3733), 20 U/µl 1.5 μl (30 units) 1.5 μl (30 units)
Nuclease-free H2O (NEB #B1500) to 25 µl 19.5 μl

* or user provided 

(1) Destination vector must possess BsaI-HFv2 restriction sites at both ends of the insert sequence and in the proper orientation.
(2) Precloned inserts must possess 5 ́ flanking bases (6 recommended) and BsaI-HFv2 restriction sites at both ends of the
inserts and in the proper orientation.

Reaction Assembly Protocol
SUGGESTED ASSEMBLY PROTOCOL 
(37°C, 5 min → 16°C, 5 min) x 30 → 60°C, 5 min → 4°C(3)

(3) If reactions are done overnight, add a 4°C terminal hold to the protocol, but repeat the final 5 min 60°C step the next day before
the transformations.

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 LB agar plates containing chloramphenicol (for pGGAselect) at 37°C for 15 min.
  2. Mix the cells thoroughly by flicking the tube and inverting, then spread 100 µl outgrowth onto each plate.
  3. Incubate the plates overnight at 37°C, or 24 hrs at 30°C, or 48 hrs at 25°C.


Twenty-four fragment assemblies of the lacI/lacZ cassette were performed using the protocol included in this article. While 30 cycles is sufficient to achieve 24 fragment assemblies, the stability of the BsaI-HFv2 and T4 DNA Ligase allows continued assembly through 45 and 60 cycles with a low background. (a) Efficiency of assembly and (b) accuracy of assembly versus cycle number. This continued functionality past the traditional 30 cycles of assembly indicates a high level of enzyme stability.


References