Quantifying gel purification products - (May/14/2007 )
QUOTE (olywa510 @ May 14 2007, 11:29 AM)
The cloning itself does not seem to be working, and I think it may have to do with insert:plasmid concentration.
Is there a way to quantify the samples without using most of it?
I have tried using a spec. but I think the TAE messes with it too much for the data to be usefull (even in terms of just builing ratios)
Is there a way to quantify the samples without using most of it?
I have tried using a spec. but I think the TAE messes with it too much for the data to be usefull (even in terms of just builing ratios)
Before I do my ligations, I usually run 5ul of my 50ul restriction digests on a gel along with 5ul of a 10x diluted 1ug/ul 1kb DNA ladder. I compare the amount of DNA in each well with the amount in the ladder, which should be 0.5ug, and then calculate the concentration of the digested vector and insert.
I think what you mainly have to worry about with ligation reactions is to avoid a high DNA concentration. If you are using NEB's T4 DNA ligase, they recomend to keep the overall concentration of vector + insert between 1-10 μg/ml for efficient ligation.
From what I have read, it seems that the insert:vector ratio is not terribly important. Is this true? I just did some ligations with 1:1, 1:3, and 1:5 vector:insert ratios, and all the plates had a comparable number of colonies.
-alexdbrot-
it depends on the fragments involved. Depending on the lenght and sequence composition, DNA can naturally be bent or straight. It might be rigid or have some degree of flex. All this affect the energetics of a DNA strand circularising, which in turn affects wherether or not we see any colonies after the ligation.
Thus some fragments ligate well, some don't. Those that don't need a little push from probability. Also once a strand becomes too long it is hard for the ends of the molecule to randomly meet and circulise. Thus more DNA is required.
-perneseblue-
QUOTE (perneseblue @ Jun 6 2007, 05:56 PM)
it depends on the fragments involved. Depending on the lenght and sequence composition, DNA can naturally be bent or straight. It might be rigid or have some degree of flex. All this affect the energetics of a DNA strand circularising, which in turn affects wherether or not we see any colonies after the ligation.
Thus some fragments ligate well, some don't. Those that don't need a little push from probability. Also once a strand becomes too long it is hard for the ends of the molecule to randomly meet and circulise. Thus more DNA is required.
Thus some fragments ligate well, some don't. Those that don't need a little push from probability. Also once a strand becomes too long it is hard for the ends of the molecule to randomly meet and circulise. Thus more DNA is required.
Yeah that had definitely crossed my mind. It facinates me how energetics, kinetics, entropy and a myriad of physics comes into play with something as simple as ends meeting in a ligation reaction.
oh how I love science. snort.
-alexdbrot-