This is the first time I am trying to clone. I am not sure where to start as I have some questions and concerns regarding the process.
I have cDNA for alpha-integrin fused with mCherry, which I want to amplify, and later do a lentiviral transduction into mammalian cells. I was told that PCR will not work, as this cDNA is too long for it. I was advised to clone instead. This is where I would highly appreciate your help. This cDNA was requested from a different university and I don't have the sequence for it. As in, I do not know if it has restriction sites at either of the ends. I understand the following steps.
- Pick a plasmid vector and insert the cDNA into it.
- Transfer it into E.Coli
- Pick the colonies which have transformed.
- Isolate cDNA.
1. As I do not have the cDNA sequence, is it still possible to attach linkers, to be able to insert it into a vector? Or which is the best way to do this?
2. How to chose the best vector plasmid? Is it alright use a commercially available kit?
3. After picking the colonies how to isolate the cDNA constricts that are now more in number?
I have read lots of protocols, but they all talk about synthesis of cDNA or library construction. As I am new to this, I am not able to make the right call. Your advice and guidance will be highly appreciated.