Jan 09, 2026
Cloning with restriction enzymes protocol
- Julia Heiby1
- 1FLI Leibniz Institute on Aging
Protocol Citation: Julia Heiby 2026. Cloning with restriction enzymes protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5krog47/v1
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: August 22, 2024
Last Modified: January 09, 2026
Protocol Integer ID: 106178
Keywords: using restriction enzyme, restriction enzymes protocol, restriction enzyme, gene fragment, gene fragment of interest, enzyme
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Abstract
How to quickly clone your gene fragment of interest into a vector, using restriction enzymes.
Materials
- Centifuge 5804R (#5805000010, Eppendorf) or 5810R (#5811000015, Eppendorf)
- Heatblock (#SBH130D3 or #SBH200D3, Stuart)
- 1.5ml tubes (Eppendorf, #0030 120.086)
- Serologicla Pipettes (Greiner, cellstar)
- Milli-Q water system (Merck, Advantage A10)
- E.coli DH5𝞪 (Thermo Fisher, EC0112)
- XhoI (Biolabs, #R01468)
- NotI (Biolabs, #R3189L)
- T4 ligase (Biolabs, #M0202T)
- agarose (Roth, #22675)
- Gel extraction kit (QIAEX®II-QIAGEN, #20021)
- DNA clean up kit (Zymo DNA Clean and Concentrate, #D4006)
- ice
- Tryptone (XXX)
- NaCl (XXX)
- Yeast (XXX)
- Ampicillin (1XX)
- Agar (XXX)
- Mini-Prep kit for DNA isolation (QIAprep®spin, 27106)
Troubleshooting
Generating Gene Fragment and cutting vector
Restriction Enzymes depending on your choice, here for example XhoI and NotI (both Biolabs, R01468 and R3189L , respectively), digesting gene fragments for 3h 37°C, or following the enzyme manufacturer's instruction
+1µl XhoI
+1µl NotI
+2µl cut smart buffer
+ Xµl gene fragment to be cut
Up to 20µl with ddH2O
Note: If not sure which enzymes to use, check your sequence, for example with SnapGene
DNA clean up
Either run the digested fragment on 2% agarose gel (Roth, 22675), cut out bands and use a gel extraction kit (QIAEX®II-QIAGEN, 20021),
or, use directly DNA clean up kit by for example Zymo DNA Clean and Concentrate (D4006) and follow manufacturer's instruction
Ligation
Ligation with T4 ligase (Biolabs) at 16°C o/n
+ 2µl T4 ligase buffer
+ 1µl T4 ligase
+ Xµl cut gene fragment
+ Xµl cut vector
Up to 20µl with ddH2O
Note: to calculate the ratio needed between gene fragment and vector, we recommend using the NEB® Calculator, as the amount required will vary depending on the length of gene fragment and vector
Transformation
Transformation into competent E.coli DH5𝞪 (Thermo Fisher, EC0112)
+ 5µl ligation sample
+ 40µl E.coli DH5𝞪
- incubate for 30min on ice
- heat shock at 42°C for 1min
- 5min on ice
- 1h in LB media 1ml at 37°C
- centrifuge and discard ~900µl of supernatent
- resuspend in leftover media and plate ~50-100µl on LB-plates containing antibiotic (selected resistance of plasmid)
- incubate at 37°C o/n
Amplification
pick single colonies from plate and transfer to 5ml LB-medium containing antibiotic of choice
incubate at 37°C on shaker o/n
Note:
LB-medium composition: MilliQ H2O (filled to 1l, and adjusted pH 7.4), Tryptone (10g), NaCl (10g) and Yeast (5g); autoclaved and added for example Ampicillin (100mg/ml stock, add 1ml to 1l LB-medium) when broth is cooled to ~50°C;
Same composition for LB-plates, with addition of agar (15g for 1l) before autoclaving
DNA extraction
use Mini-Prep kit for DNA isolation following the manufacturers instructions (QIAprep®spin, 27106)
Sequence validation
Sequence plasmid with appropriate primers and check sequencing results