cDNA library insert-vector ratio - (Jun/25/2007 )

Hi All

My name is Bob.

I have a problem with calculating insert-vector ratio, but not because I do not know the equation, but because in my, let say fraction one, there is a lot of cDNA's of different sizes. So what value I should have in cDNA(insert) size place. I just started cDNA library and a pile of gold will be given to anyone who helps.

A Big Thanks in advance

P.S. I am impressed with the knowledge of all those answering questions.

Bob

Hey Bob,

I've faced this exact issue before, so (hopefully) I can offer some helpful advice.

The first thing to consider is why you're building this library. If you will eventually screen it for a specific clone with a (approximately) known length you'll have the most success finding your clone of interest if you use this length as your insert size.

If you're trying to make a comprehensive full-length library, there are two approaches I'd suggest:

1. If you're using a kit, use the average cDNA size quoted by the manufacturer as your insert length (will probably be around 2 kb for mammalian cDNAs). Just make it sure it seems a reasonable approximation based on your actual cDNA lengths. This will bias against longer cDNAs, but still gives reasonable results. (In my experience, the average insert size in my libraries was always shorter than the average cDNA length quoted by the manufacturer, but still closer to 2 kb than 1 kb.)

2. To really maximize the recovery of every clone, you could do a series of separate ligations with various cDNA size fractions. I don't know what your exact protocol is to make cDNA, but I have used the Superscript Plasmid system. Using this kit, following column purification you have a series of size fractionated cDNAs in separate tubes. Because the cDNA is spiked with P-32, you can estimate the size of cDNAs contained in each fraction by running a small aliquot on a gel alongside a radiolabelled DNA ladder (make sure you run an alkaline gel for accurate size estimation!) If you have sufficient cDNA amounts, you could set up a few different ligations with different amounts of vector DNA to have a more uniform insert:DNA ratio across the entire library. This would increase the overall coverage of your library, but does take more work, and more competent cells. (=more $$$)

A word of caution: try to minimise the amount of library amplification following transformation. Propagating a library in E.coli to 'get more clones' actually achieves the opposite effect: shorter cDNA inserts become even more overabundant, thereby making rare or long inserts even harder to find in a screen.

Also, it goes without saying that for a library, you really really really need a good vector that gives no background. If you're making your own, you should check it beforehand using a positive control (an insert with the same sticky ends as your eventual cDNAs and of reasonably long length (at least 2kb)) and a negative control with no insert. Unless the difference between your positive and negative control ligations is night and day (a difference measured by orders of magnitude) you'll end up with a lot of empty clones in your library.

Have fun

Ginger Spice