GHOST in Electrophoresis - (Aug/24/2010 )

YOu could try a phenol chloroform extraction, it takes some time to do but usually gives excellent results.

Take the migration of EtBr out of the equation -- run your gel without adding ethidum to the agarose, and submerge the gel in an EtBr solution after the run. Destain and photograph -- see whether the "ghosts" appear under those circumstances.

bob1 on Sun Aug 29 21:37:17 2010 said:

I have done that post extraction. Shall sit down to take its OD and will post it soon.

HomeBrew on Mon Aug 30 03:12:16 2010 said:

Thanks for the suggestion HomeBrew. Shall try it out when possible.

Ameya

bob1 on Sun Aug 29 21:37:17 2010 said:

I had performed this PCI extract quite some time ago and just yesterday could get spectrophotometer readings.

So, after treatment, one of my samples gives readings of ~0.5 at 260 and 280 nm (the ratio still being lesser than 1). The other one (tested within minutes after the first one and using the same blank as before) gave negative readings at both the wavelengths.

We do not use the spec. very frequently, but I have also seen that I cannot set the blank to zero at wavelengths such 230 and 320 nm. Does anyone know why it is so????
(Note: It is a manual spectrophotometer)


Thanks

I usually run a UV profile instead of single read at specific wavelengths. That way I can see if the readings are blanked correctly. What cuvettes are you using? Some materials can't be used at 230nm. Not sure what's the problem with 320nm though 'cos most that's transparent for most cuvettes.

donny on Sat Sep 18 10:38:02 2010 said:

I am using quartz cuvettes. I have taken readings at 320nm before, but now I cant set my blank to zero anymore at that wavelength.

Perhaps there's something wrong with your spec? If so, none of the readings can be trusted...

Make a DNA sample of known concentration using calf thymus or salmon sperm DNA and see if the reading matches your known concentration.

HomeBrew on Mon Sep 20 12:07:42 2010 said:

Is there something more home-made that I could use. Calf-thymus and salmon sperm are not available in my lab. We deal with human blood samples here.

You should be able to buy either of them in lyophilized form in more than sufficient quantities for around $40.00 US. Since it seems you use your spec often, having a known standard would not be a bad investment.

The "homemade way" would be to make a series of dilutions of a quantity of DNA you've previously recovered that showed a high spec reading (assuming that means there's a reasonably high concentration of DNA in the sample). You can run such a series on an agarose gel along with a DNA ladder wherein the concentration of the DNA in the ladder is known, and stain the gel with ethidium bromide. Ethidium bromide fluoresces in proportion to DNA concentration, so you can compare the bands of your dilution series to that of the ladder of known concentration -- the dilution that flourishes about as brightly as the ladder bands is about the same concentration as the ladder. You can then use this sample as a known value to test your spec.

You could also just dilute your DNA ladder to a known concentration, and use that.