VPS13D DNA plasmid generation
Marianna Leonzino, Andrés Guillén-Samander, Pietro De Camilli
Published: 2021-08-18 DOI: 10.17504/protocols.io.bvgwn3xe
Abstract
This protocol describes the basic molecular cloning technique utilized for the generation of VPS13D constructs in [https://doi.org/10.1083/jcb.202010004](https://doi.org/10.1083/jcb.202010004). This protocol and the enzymes included in it are commercialized by Takara Bio.
Due to low expression of big DNA constructs (>10kb), we decided to generate a codon-optimized cDNA encoding for human VPS13D, including an mScarlet fluorescent protein after aminoacid 1576 flanked by BamHI restriction enzyme sites. The construct was generated by and purchased from Genscript. From this initial construct, we generated several VPS13D constructs. The specific primers and enzymes used for each construct are included in Table 1 of our manuscript: [https://doi.org/10.1083/jcb.202010004](https://doi.org/10.1083/jcb.202010004)" . For most of our cloning procedures, Infusion cloning (Takara) was used.
Attachments
Steps
VPS13D DNA plasmid generation
1.
Linearize the backbone with restriction enzymes or by PCR amplification.
2. Suggestion : Takara has an online tool to help with primer design at: Suggestion: Takara has an online tool to help with primer design at: https://go.shr.lc/3daJf0E
Amplify the insert by PCR with primers including 15nt of overlapping sequence with the target backbone.
Note
3.
Run both products in an agarose gel to confirm expected size and purify the DNA from gel using a NucleoSpin Gel and PCR Clean-up kit (Takara).
4. Suggestion : We found that big DNA constructs accumulate mutations easily when amplified in bacteria, this can be minimized by growing bacteria at 30°C.
Ligate the linearized backbone and the insert with the Infusion enzyme mix (Takara).
Note
