Minimum sample required for visualization on gel - How to load 1ul of DNA on gel (Aug/15/2006 )

I gel extracted a restrction fragment and want to use it for cloning in two vectors, one for sequencing to check I got the right fragment and the other for using it in my experiment if the sequence is correct. So I need a lot of this fragment. so I do not want to waste any. The qiagen kit for extraction suggests to load 1ul on gel, it also gives weight and conc. comparison charts for 1ul loads.

So what volume of gel do I prepare, and how much 10X loading dye do I add to 1ul of sample? Do I need to do anything to make 1 ul sample more visible on the gel, like add more EB?

Thanks

Hi nifT!

Loading 1 µl is no problem if the concentration is high enough...
but if you use 10x loading buffer I would suggest to mix your sample with water, pipetting 0.1µl might be too difficult...
If the concentration could be too low, I would also use the smallest slot size to prepare the gel, because then the DNA is more focused and easier to see.
If you don't see anything you can try incubating the gel in more EB...
In my group we usually prepare our gels with buffer that already contains EB - and I am able to see 1µl... so I don't know if there is a special volume to prepare - but I guess, the thinner it is, the easier it is to stain...

But maybe you have the possibilty to use a Nanodrop in your or a neighbour group? You only need 1µl and get the exact concentration.

hm, what else? Maybe: Just try and good luck!

Chakchel

what's the composition of your 10x loading buffer? if it's only dye and glycerol, then there is no problem. just mix 1µl sample and 1µl loading buffer, which you can predilute to 2x with water.
even if you don't do that, there should be no problem, as you'll have a higher glycerol concentration. this is nothing to be worried about, as the sample will be just more dense, but on the gel it'll run as expected anyway.

I would not load 2µL on a gel, because you lose a little of sample when your tip touch the running buffer. I would load 1µL of sample + 1µL of 10x loading dye + 8µL water.
(you lose the same little volume, but not the same amount of your sample, only 10 times less)