DNA quantification after gel purification - (Sep/08/2010 )

I have just done a gel purification of my digested vector and insert and wanted to check the concentration of them to make sure it was what I thought it was, but when I placed 2ul of the final flowthrough of the gel purification column onto the nanodrop spectrophotometer I could not make out any DNA at all. The graph just dropped off rapidly to zero and had no wave to it.

Does gel purification interfere with the spectropic properties of DNA in any way?

I am doing a ligation at the moment based on estimated DNA amounts given the amount of DNA I started with before the digest and hope to transform later, so if I get colonies then I suppose I will know that I had enough DNA.

During gel purification you would lose a lot of DNA. that is very normal. You just need to run a lot of sample on the gel, cut a big fragment, and gel purify to be able to get at least 10ng by Nanodrop. Also let the water remain in the column for 1-5 minute before the last centrifugation step.

I´d say a far more reliable (and old fashioned) way to quantify a PCR product is to run your gel-purified DNA on a second agarose gel (small wells). The intensity of your band of interest can then be compared to the intensitiy of one of the fragments in the DNA ladder. DNA ladders come with a description of the conc. of each band in the ladder. If you you add to it a certain volume of loading dye and know the volume you load on the gel, you know exactly how many ng of DNA is in a specific band.

Well I did my ligation and transofrmation yesterday anyway and I have colonies on my Amp plates now so there must have been some DNA there in the Vector sample anyway!

The fact I have roughly the same number of colonies on my vector + no-insert control plate as on my Vector + insert plate is still a worry though, especially since I used TSAP alkaline phosphatase so the vector shouldn't re-ligate to itself at all! Unsure if it is worth doing minipreps to check if the insert is there or whether should just assume the insert didn't ligate.

I had totally opposite experience from yours.

I found that Nanodrop always gave me high DNA concentration reading for my gel purified product. When I ran the DNA on an agarose gel, the thickness of the band did not match the reading from Nanodrop. I am interested to know the reason, if anyone can explain it.

Anyway, when I set up my ligation reaction, I never bother about the DNA concentration. I always play with the vector and insert ratio, for example 1 ul of vector DNA and 2 ul of insert DNA, and I still get my constructs, lots of them are sitting in the freezer now. Of course, my current supervisor dislikes the way I do my cloning, but I don't care. Sometimes science can be a little less scientific. haha...

tats cool, i never bothered abt vector insert ratio, jus i ll run a gel to check whether i retained any vector and insert after gel purification, if i see bands in both jus i ll add assuming 1 times vector and 3 times insert, in my experience i would say one neednt have to worry abt anything in cloning unless ur competent cells are good enough...

gnana...

virusfan on Fri Sep 17 15:38:04 2010 said:

Nanodrop measures the absorbtion of your sample at 260 and 280 nm (and 320?), not specificaly the concentration of DNA, it's only extrapolated that if the sample contains 100% of DNA, its concentration would be the value your Nanodrop shows. But there are other things that can absorb in the same wavelength and skew the measuring - RNA, protein, salts, some chemicals from the isolation kit and so on. The 260/280 (and 260/320) ratio should tell you roughly how pure your DNA is, because those other contaminants absorb differently in 280 (320) nm.
The gel with EtBr on the other hand shows you only the dsDNA that's there, it may tell you more about the DNA of your interest when you have a sample with low purity.