Jan 22, 2020
Nested Gibson Assembly
- 1J. Craig Venter Institute;
- 2J. Craig Venter Institute, Synthetic Biology & Bioenergy Group
- Protist Research to Optimize Tools in Genetics (PROT-G)
- JCVI West Protocols
Protocol Citation: Erin Garza, Vincent A Bielinski 2020. Nested Gibson Assembly. protocols.io https://dx.doi.org/10.17504/protocols.io.bbikikcw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 17, 2020
Last Modified: January 22, 2020
Protocol Integer ID: 32044
Keywords: Gibson, nested
Abstract
This method can be used to increase the efficiency of Gibson Assemblies containing many pieces and/or difficult to assemble DNA fragments.
Guidelines
Use this protocol if your Gibson Assembly repeatedly fails. This method will decrease the amount of DNA pieces going into your Gibson, thereby increasing efficiency of assemblies originally containing a lot of pieces. This protocol has also been used to successfully get colonies from Gibson Assemblies that contained difficult to work with DNA (i.e. AT-rich fragments or significant amounts of secondary structure in certain DNA pieces). Even if a Gibson Assembly failed overall, some of the DNA pieces in the reaction may still be ligated together. By PCR amplifying these already assembled fragments you can then repeat the Gibson Assembly with less pieces.
Materials
MATERIALS
Gibson Assembly Master Mix - 50 rxnsNew England BiolabsCatalog #E2611L
Zymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
PrimeSTAR Max DNA PolymeraseTakara Bio Inc.Catalog #R045A
NEB 5-alpha Competent E. coli cellsNew England BiolabsCatalog #C2987H
Thermocycler
Agarose
Gel box
UV imager
Waterbath
Incubator
LB broth and plates with antibiotics
Before start
If your transformation of a Gibson assembly failed to yield any colonies or testing revealed that your assembled plasmids are incomplete, then save the remainder of your Gibson Assembly. The Gibson Assembly will function as template for PCR.
Select primers
Select primers
Using the same primers that you used to amplify your DNA pieces for the Gibson Assembly, you can pick new primer pairs to amplify multiple fragments that have already been ligated together from the failed Gibson reaction.
Example: By using the A-Fwd and B-Rev primers in a PCR reaction, fragments A and B can be amplified as one piece. The C-Fwd and E-Rev primers can also be used to amplify fragments C, D, and E as a single piece. The Gibson Assembly can then be repeated with a total of 3 fragments instead of 6.
Fragment- 1. A-B
2. C-E
3. F
Example of a 6-piece Gibson Assembly being amplified to reduce the total number of pieces to 3
PCR
PCR
Run the PCR reactions using the chosen primer pairs from step 1 and the failed Gibson Assembly as the template.
Reaction Mixture
25 μl PrimeSTAR Max DNA mastermix
22 μl DI water
1 μl Forward primer [10 μM]
1 μl Reverse primer [10 μM]
1 μl Failed Gibson Reaction
------------------------------------------------
50 μl Total
Cycling Conditions
- 98°C for 10 sec.
- Tm-5°C for 15 sec.
- 72°C for 10 sec./kb
* Run steps 1-3 for 30 cycles
Run a gel
Run a gel
Run the PCR fragments amplified from step 2 on a .8% - 1% agarose gel to ensure that each of the reactions was successful.
Note: If a PCR reaction of some of the pieces failed, then repeat step 1 with different primer pairs to amplify a different set of fragments together. This also acts as a diagnostic tool to determine which DNA pieces are not assembling efficiently.
Purify DNA
Purify DNA
Purify the remainder of each of the PCR reactions. We use a Zymo Research DNA Clean and Concentrator kit.
Gibson Assembly
Gibson Assembly
Set up Gibson Assembly in a PCR tube
1 μl each DNA fragment (we dilute each of our fragments to 30 fmol)
5 μl Gibson Assembly Master mix (NEB)
x μl DI water
--------------------
10 μl total
*Incubate the Gibson Assembly in a thermocycler at 50°C for 1 h.
Transform
Transform
Mix 5 μl Gibson Assembly with 25 μl NEB 5-alpha chemically competent cells and hold on ice 5-10 min
Heat shock cells at 45°C for 30 sec and immediately place back on ice for 5 min
Add 270 μl SOC to the cells and incubate at 37°C for 1 h
Plate 50 μl cells on an LB plate containing the appropriate antibiotics and grow overnight at 37°C
Verify Transformants
Verify Transformants
Grow resulting colonies in LB broth + antibiotics and perform colony PCR, restriction digestions, and/or sequencing to verify that the assembled plasmids are correct.