Cloning cDNA construct into E.Coli. - (Jun/11/2013 )
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.
So my questions are:
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.
I think you are a little confused as to your terminology - cDNA is copy or complement DNA that is derived from RNA, it can be used for a number of things including cloning. Plasmids may contain a cDNA sequence, but they are not cDNA themselves, they are a form of double stranded DNA which can be used to obtain cDNA if a few requirements are met.
Incidentally it is unlikely that PCR is not possible on your gene, unless it is high G/C content or contains lots of repeats. It is possible (not easy though) to do PCR up to about 20 kbp (20,000 base pairs), which is much larger than the majority of genes.
It is unlikely but not impossible that you will get cDNA from the other university, it is more likely that they have cloned it into a vector (maybe without the mCherry) and your job will be to subclone it into your vector. The people that give you the DNA should tell you the vector and which site(s) it has been cloned into, but if they don't tell you the sites (or the vector), you can sequence from parts of the gene of interest to find out what sites are available and which vector they used.
If you only get cDNA, it is definitely possible to clone it into a vector - you first need to convert it into dsDNA, and then you could use blunt end cloning to clone at a single site, or as you mentioned, use adapters to add suitable sites. You could even add a single T overhang at each end and do T/A cloning.
The choice of vector depends on what you want to do with it - as you are wanting to do some lentiviral transduction, there will be specialist plasmids required for it.
After picking the colonies, you will grow up the plasmid in a broth culture, make a glycerol stock, and do a prep - these are, depending on scale, usually called something like "miniprep" or "midiprep" and are designed to isolate the plasmid specifically. You should then sequence to determine if the insert is correct. To get mRNA from a plasmid requires that the plasmid has an appropriate promoter which can be used to transcribe the DNA sequence and then turn that into cDNA.
If you can find it, Molecular cloning: a laboratory manual, by Sambrook et al., will be a very helpful resource for you (it is the cloning bible) - another useful resource is the protocols page at openwetware.
Thank you for your detailed reply. I talked to the other university and they told me that it is cDNA and sent me the entire sequence for it.
Now, I am wondering if there is a way to find out the ratio of GC to AT content, so that I can rule out on PCR. Also, I am planning in lentiviral transduction in later stages of experiment, but does that affect the choice of plasmid in cloning of this mCherry-integrin construct? In this case, could you give me some examples of the kinds of plasmids I need to look for?
Thanking you in anticipation!
Heres a G/C content calulator.
I don't know much about lentiviral production, but you should be able to find some good guides on the web.
We're using pRRL backbone vector for lentiviral transduction into HEK293T cells and it workes quite fine. Maybe someone at your institute can provide you with that?