Our modified SDM method (https://rdcu.be/dACZZ) has no limitation for deletion and been used by many researchers with high success rate. No expensive kit required, what you need are a proofreading thermal polymerase, DpnI, and normal PCR regents.

The attached file is my easy-follow lab protocol summarised based on that publication.

Best wishes and good luck.

I agree with Huanting Liu

Kits are nice for convenience, but the process is straightforward.

You just need the appropriate primers to perform the inverse PCR with a proofreading polymerase and then DpnI to remove template and kinase/ligase to close the system.

Dear Sandra Wong

you can simply do it using the PIPE cloning approach.

you can find more information about it in following papers

Article The Polymerase Incomplete Primer Extension (PIPE) Method App...

Article Combining the polymerase incomplete primer extension method ...

as well as in the following link on my blog, ProteoCool

https://www.blogger.com/video.g?token=AD6v5dx7hIjOrRfLt70Lph2VJXXgyfu7CVeVUqMIzPNb5ySq3fHJF5QSXLTq93C-8iRHI_fT9_JfcwYQiGCHj0uYBMQzkB7DU8_qi34YSr9ZJzA7OYfHQav5-9eO6Idz5f06bydovl8

https://proteocool.blogspot.com/2021/09/tips-of-week12-rapid.html

good luck

Manuele

Hi Sandra Wong ,

as has been already mentioned, you do not need kits. But I would go even further and say that you do not need even DpnI. Using PCR with primers with homologous ends, you can easily assemble the whole plasmid with your insert and if you do 2 consecutive PCRs, you can dilute the template DNA and thus remove the need for DpnI treatment.

If you want, I can help you with designing the primers and set up the protocol.

Article In vivo cloning of up to 16 kb plasmids in E. coli is as simple as PCR

The Method I like is inverse PCR with 5'- phosphorylated forward and 5'-phosphorylated reverse primers facing away from each other and blunt end ligation with T4 DNA ligase and ATP and magnesium.

https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/site-directed-mutagenesis-platinum-superfi-app-note.pdf

I linked article, the method is Mutagenesis protocol B with deletion:

Note: the primers must be 5'-phosphorylated. Primers are not 5'-phosphorylated unless you order them from the manufacturer as phosphorylated. Otherwise, you can phosphorylate primers with T4 Polynucleotide kinase and ATP and magnesium. Then ligate the phosphorylated PCR product with T4 DNA ligase and ATP and magnesium.

I use Phusion DNA polymerase for this. Any thermoresistant high fidelity DNA polymerase should work except Taq DNA because (because Taq creates PCR products with 3' overhangs containing one extra overhanging A base. That is why Taq is used for TA cloning.)