Cloning – Quick Instructions
These instructions are provided as a quick reference for users experienced with molecular biology techniques and cloning. For complete instructions please refer to the complete user guide or Sambrook J and Russell DW, editors. (2001) [1]
Cohesive-End Cloning
Cohesive-end cloning is highly efficient due to the hydrogen bond stabilization of the complementary DNA overhangs that are created by many restriction digests.
Required:
- gBlocks Gene Fragments with non-phosphorylated ends and include the restriction sites at least 5 bases internal from the ends
- Vector containing the appropriate restriction site(s)
- Desired restriction endonuclease(s)
- Alkaline phosphatase
- T4 DNA Ligase
- Competent cells
Procedure:
- Linearize 1 μg of vector by restriction digest
- Remove the 5’ phosphates from the vector with an alkaline phosphatase
- Gel purify the linearized vector
- Resuspend the gBlocks Gene Fragments in 20 μl of TE (10 mM Tris, 1mM EDTA, pH 7.5)
- Prepare ends of gBlocks Gene Fragment by restriction digest of 10μl
- If chosen restriction enzyme cannot be heat inactivated, column purify and elute in 10-20 μl following manufacturer’s instructions
- Place the following in the ligation reaction:
- 80 ng gBlocks Gene Fragments
- 200 ng of vector from step 3
- 400 cohesive end units of T4 DNA ligase
- Fresh T4 DNA ligase buffer diluted to 1X
- Total volume 20 μl
- Incubate for 2 hours at 16°C
- Transform 2 μl onto competent cells following the manufacturer’s instructions
Preparing gBlocks Gene Fragments for TA cloning
TA cloning is a subtype of cohesive end cloning that is fast and highly efficient for non-directional cloning of DNA. It takes advantage of the residual adenine residue that is typically added on the 3’ end of DNA fragments by the terminal transferase activity of enzymes, including Taq DNA polymerase. The resulting end is compatible with TA-cloning vectors that can be purchased from several manufacturers.
Procedure:
- Resuspend the gBlocks Gene Fragments in 20 μl TE (10 mM tris, 1mM EDTA, pH 7.5)
- Add 5 μl of the diluted gBlocks Gene Fragments to a 15 μl mixture containing the following:
- 1-3 Units Taq polymerase
- 0.05 mM dATP
- 1X Taq polymerase Buffer
- 1.5 mM MgCl
- Incubate the mixture at 70°C for 15-30 minutes and use 1-10 μl for TA cloning following the manufacturer’s instructions.
Blunt-End Cloning
Blunt-end cloning is also a popular cloning method. However, its efficiency is typically 50-100 fold lower than cohesive end cloning, and is non-directional [1]. Due to the poor efficiency and limited starting material, we do not generally recommend blunt-end cloning of gBlocks Gene Fragments by non-experienced users. Users that wish to use this method are advised to amplify the gBlocks Gene Fragments with 5’ phosphorylated primers to ensure there is enough material for a repeat of the cloning if needed.
Required:
- gBlocks Gene Fragments with phosphorylated ends
- Vector containing the appropriate restriction site
- Desired restriction endonuclease (RE)
- Alkaline phosphatase
- T4 DNA Ligase
- Competent cells
Procedure:
- Linearize 1 μg of vector using a blunt-cutting RE such as EcoRV or MscI
- Remove the 5’ phosphates from the vector with an alkaline phosphatase
- Gel purify the linear fragment of the vector digest
- Resuspend the gBlocks Gene Fragments in 20 μl of TE (10 mM Tris, 1mM EDTA, pH 7.5)
- Place the following in the ligation reaction:
- 8 μl gBlocks Gene Fragments (80 ng)
- 200 μg of vector from step 3
- 400 cohesive end units of T4 ligase
- Fresh T4 DNA ligase buffer diluted to 1X
- Total volume 20 μl
- Incubate for 2 hours at 16°C
- Transform 2 μl onto competent cells following the manufacturer’s instructions
References
- Sambrook J and Russell DW, editors. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory
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