Mar 01, 2025

Public workspaceProtocol for Gibson Assembly (PrimeStar + In-Fusion) V.2

DOI
dx.doi.org/10.17504/protocols.io.81wgbrknnlpk/v2
  • 1Washington University
Carolina Lopez
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.81wgbrknnlpk/v2
Protocol Citation: Carolina Lopez 2025. Protocol for Gibson Assembly (PrimeStar + In-Fusion). protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbrknnlpk/v2Version created by Carolina Lopez
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: February 02, 2025
Last Modified: March 01, 2025
Protocol Integer ID: 120019
Abstract
Procedure for cloning using Gibson Assembly using PrimeStar and In-Fusion kits
Materials
  • Plasmids
  • DNA template
  • Restriction Enzymes (New England Biolabs)
  • 10x Cutsmart Buffer (New England Biolabs)
  • PrimeSTAR Max DNA Polymerase (TaKaRa, #R045A)
  • Agarose
  • EtBr
  • NEB Monarch DNA Gel Extraction kit (NEB #T1020)
  • In-Fusion Snap Assembly Master Mix (TaKaRa, #638947)
  • TOP10 cells

Vector digestion
Vector digestion
30m
30m
1. Double digest Vector with New England Biolabs Restriction Enzymes: 80uL reaction

AB
ComponentVolume (uL)
DNA plasmidX ul for 4 ug of plasmid
10x Cutsmart Buffer8 uL
Enzyme 14 uL
Enzyme 24 uL
NucleaseFree H2O64-X uL
2. Incubate for Duration00:30:00 at Temperature37 °C .

Notes:
  • Not all two enzymes can be used together in a double digestion. Think about it ahead and you can test your two enzymes here: https://nebcloner.neb.com/#!/redigest.
  • Enzymes are different, but most can be digested in CutSmart buffer at 37°C within 30 minutes. This is a general protocol. Please check the best digestion conditions for your enzymes in this web：https://nebcloner.neb.com/#!/redigest. It will provide the temperature, buffer, and incubation time specific to the two enzymes you are using.
30m
Insert fragment PCR with PrimeSTAR Max DNA polymerase
Insert fragment PCR with PrimeSTAR Max DNA polymerase
10m 30s
10m 30s
Set up PCR Reaction (50uL):
AB
PrimeStar Mix (2X)25 uL
F primer1 uL
R Primer1 uL
DNA template20-50 ng
H20up to 50uL

Run PCR Conditions:
a) Temperature98 °C Duration00:00:10
b) Temperature98 °C Duration00:00:10
c) Temperature55 °C Duration00:00:10
d) Temperature72 °C X seconds
e) Repeat step b-d 30 times
f) Temperature72 °C Duration00:10:00
g) Temperature4 °C hold

Note:
  • DpnI treatment after PCR is recommended for same antibiotic-resistant backbone: Add 1 µL of DpnI to a 50 µL PCR reaction and incubate at 37°C for 1 hour.
  • You might to increase the amount of DNA template if your template is cDNA.
  • X depends on the length of your PCR product, 15s per kb.
  • Step C: Using 55°C has worked perfectly for me for over 10 years with PrimeSTAR DNA polymerase. However, the success also depends on how your primers are designed.
10m 30s
Run DNA gel
Run DNA gel
40m
40m
  1. Add 6x loadig Dye to digestion and PCR reaction and vortex briefly to mix.
  2. Make 1% agarose gel.
a) Mix 1 g of Agar with Amount100 mL of TAE Buffer.
b) Microwave to boil agarose and let cool until you can touch bottle, but gel is not solid.
c) Add Amount2 µL of EtBr to agarose and pour into DNA gel mold with 10 well comb.
d) Let gel solidify.
3. Load Amount100 µL of reaction into well of gel
4. Run gel for Duration00:40:00 at 120V.
5. Visualize band with UV light and cut out section of gel with band with new razor blade and place in Amount1.5 mL tube.
40m
Purification DNA from gel with Monarch DNA Gel Extraction kit
Purification DNA from gel with Monarch DNA Gel Extraction kit
  1. Weigh the gel slice.
  2. Add 4 volumes of Monarch Gel Dissolving Buffer to the tube with the gel slice (e.g., 400 μl buffer per 100 mg agarose). If the gel slice is >150 mg, consider reducing the amount of Gel Dissolving Buffer to 3 or 3.5 volumes to minimize the guanidine salt present in the workflow.
  3. Incubate the sample between 37–55°C (typically 50°C), inverting periodically until the gel slice is completely dissolved (generally 5–10 minutes).
  4. Insert the column into collection tube and load sample onto the column. Spin for 1 minute, then discard flow-through.
  5. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
  6. Repeat wash (Step 5).
  7. Re-spin the column and tubes.
  8. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If in doubt, re-spin for 1 minute before placing into clean microfuge tube.
  9. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, and spin for 1 minute to elute DNA.

Notes:
  • Step2: If the volume of the dissolved sample exceeds 800 μl, the loading of the sample onto the column should be performed in multiple rounds to not exceed the volume constraints of the spin column.
  • Step3: For DNA fragments > 8 kb, an additional 1.5 volumes of water should be added after the slice is dissolved to mitigate the tighter binding of larger pieces of DNA (e.g., 100 mg gel slice: 400 μl Gel Dissolving Buffer: 150 μl water). Failure to dissolve all the agarose will decrease the recovery yield due to incomplete extraction of the DNA and potential clogging of the column by particles of agarose.
  • For PCR products, if you have only one specific band, you can skip the gel running step and purify DNA directly from the PCR products.
Gibson Assembly with TAKARA In-Fusion
Gibson Assembly with TAKARA In-Fusion
1h 48m 30s
1h 48m 30s
1. Calculate the molar ratios of Vector and PCR product used
a) https://nebiocalculator.neb.com/#!/ligation
b) I usually use 1:1 or 1:2 ratio, and the vector amount I use is 50-60ng


2. Mix Mater Mix, vector, insert and H20 together.
3. Incubate for Duration00:15:00 at Temperature50 °C .
4. Transform Product into E. coli
a) Add Amount2 µL of product to Amount50 µL of TOP10 cells.
b) Incubate for Duration00:30:00 on ice.
c) Heat shock bacteria in Temperature42 °C waterbath for Duration00:00:30 .
d) Incubate on ice for Duration00:03:00 .
e) Add Amount500 µL of SOC media and shake in warm room for Duration01:00:00 .
f) Spin down the bacteria and discard the supernatant, leaving only 50-100 µL. Then, resuspend the bacteria in the remaining volume.
g) Plate bacteria onto LB-Antibiotic Plate. And incubate overnight in Temperature37 °C warm room.

Note:
  • Please read the In-Fusion Snap Assembly User Manual before your start.
  • This kit allows the insertion of more than one fragment; refer to the User Manual for detailed instructions.
  • Incubation time at 50℃ could be extend to 30min, but longer time may not that helpful, think about other things if you didn't get clones.
  • The incubation time at 50°C can be extended to 30 minutes if you have more than one insertion or the plasmids is big, but longer durations may not significantly improve results. If you do not get clones, consider troubleshooting other things of your experiment.
1h 48m 30s
Protocol references