Gibson Assembly: Difference between revisions

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=Protocol=
=Protocol=
==Materials==
*DNA fragments for assembly:
**50-100 ng of base vector fragments
**2- to 5-fold molar excess of insert (smaller fragment = higher molarity needed)
**'''15-500 base pairs of homology at ends of fragments'''
*Gibson Assembly Master Mix
**2xNEBuilder HiFi from NEB
**1.33xGibson Assembly Master Mix, made from components in lab (see below)


==Recipe for Gibson Assembly Master Mix==
''5X Reaction Buffer''


To a 15 mL tube, add:
1. Prepare DNA required for the reaction, typically linearize vector (see restriction digest) and PCR amplification of insert(s) (see PCR)
*1.5 g PEG-8000
*363.1 mg Tris-HCl
*46.3 mg DTT
*19.9 mg β-NAD
*600 μL 10mM dNTP mixture
*300 μL 1M MgCl₂
Add 3-4 mL ddH₂O and vortex thoroughly. Vortex and pipette vigorously to break up PEG-8000 crystals. Use pipette to measure volume in tube, and add water to a final volume of 6 mL. Place on ice if using immediately, or store in freezer.


<pre>This reaction buffer can be stored at -20˚C for up to 6 months, or -80˚C for at least 12 months (β-NAD denaturation).
2. In a PCR tube, prepare reaction mix as indicated below
6 mL of reaction buffer 1500 x 15 μL reactions</pre>
{| class="wikitable"
|
|'''2-3 Fragments'''
|'''4-6 Fragments'''
|-
|Recommended DNA Molar Ratio (Vector:Insert)
|1:2
|1:1
|-
|Starting amount of vector
|50 – 100 ng
|50 – 100 ng
|-
|Amount of insert
| colspan="2" |depends on molar ratio
|-
|2X HiFi DNA Assembly Master Mix
|10 µL
|10 µL
|-
|ddH<sub>2</sub>O
|x (up to 20 uL)
|x (up to 20 uL)
|}
3. Incubate the sample in a thermocycler at 50°C for 15-30 mins for 2-3 fragments or 60 mins for 4-6 fragments


''1.33X - 2X Master Mix, yields thirty 15 μL aliquots''
4. Transform into component E. coli


To a pre-chilled 1.5 mL tube, add:
==Reference==
*292.3 μL ddH₂O ('''''NOTE''': to create 2x Master Mix, allowing for up to 10 μL DNA per reaction, use only 142.3 μL ddH₂O'')
NEBuilder HiFi DNA Assembly Master Mix Instruction Manual
*120 μL 5x Reaction Buffer
*0.24 μL 10U/μL T5 Exonuclease
*7.5 μL 2U/μL Phusion DNA Polymerase
*45 μL 40U/μL Taq DNA Ligase '''OR''' 15 μL 40U/μL Taq DNA Ligase + 1 μL 3000 U/μL T7 DNA Ligase + 0.6 μL 100 mM ATP + 28.4 μL ddH₂O


Aliquot in 15 μL volumes. Store at -20˚C for 3-6 months.
https://nebcloner.neb.com/#!/products/search/E2621
 
==Reaction==
If using NEBuilder HiFi:
*Add 10 μL of DNA (+ water) to 10μL of 2xNEBuilder HiFi
If using homemade Master Mix:
*Add 5 or 10 μL of DNA (+ water, depending on indicated concentration of Master Mix) directly to tube containing 15 μL aliquot of master mix
 
Incubate at 50˚C for 45-75 mins ('''if Master Mix contains T7 DNA Ligase''': incubate for additional 15-30 mins at 25˚C or room temp).<br />
Transform 1-4 μL into competent bacteria, or store mixture at -20˚C.

Latest revision as of 19:55, 17 January 2025

Gibson Assembly, also known as enzymatic or chew-back assembly, is a powerful tool used for DNA cloning. It allows you to manipulate almost any fragment of DNA in any location, alleviating the need to plan projects around restriction sites.

Overview and Background Info

Gibson Assembly uses sequence homology between fragments of DNA to join them together. A Gibson Assembly reaction mixture uses 3 enzymes: 5'-->3' exonuclease, high-fidelity polymerase and ligase. Together, these enzymes chew back the 5' end of DNA fragments (allowing fragments with sequence homology to anneal), fill in any gaps created by the chew-back, and seal the nicks in the annealed fragment. While it can be used to generate linear assemblies, Gibson is most commonly used to manipulate circular plasmids, for transformation and propagation in bacteria. Fragments for Gibson Assembly must have 15-500 bp of homology. This can be done through synthesis of entire fragments, or by amplification with "overhanging" primers

Protocol

1. Prepare DNA required for the reaction, typically linearize vector (see restriction digest) and PCR amplification of insert(s) (see PCR)

2. In a PCR tube, prepare reaction mix as indicated below

2-3 Fragments 4-6 Fragments
Recommended DNA Molar Ratio (Vector:Insert) 1:2 1:1
Starting amount of vector 50 – 100 ng 50 – 100 ng
Amount of insert depends on molar ratio
2X HiFi DNA Assembly Master Mix 10 µL 10 µL
ddH2O x (up to 20 uL) x (up to 20 uL)

3. Incubate the sample in a thermocycler at 50°C for 15-30 mins for 2-3 fragments or 60 mins for 4-6 fragments

4. Transform into component E. coli

Reference

NEBuilder HiFi DNA Assembly Master Mix Instruction Manual

https://nebcloner.neb.com/#!/products/search/E2621

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