GHOST in Electrophoresis - (Aug/24/2010 )

Hello everyone.

I use a QIAGEn Puregene Blood Core kit A for my gDNA isolation. Recently, I decided to quantify the DNA using a manual spec. for which I got OD of 0.216 (260nm) and 0.262 (280nm). I use a dilution factor of 150. I guess it means there is contamination in the DNA I obtain. I also see certain blocks move in the opposite direction of the DNA during electrophoresis (which I call GHOST) which is progressive with time and is also seen when my PCR does not work. I need advice on removing the ghost and getting better OD readings for my DNA.

Thanks

Can you post a picture of this phenomenon? It will help us to diagnose the problem.

bob1 on Tue Aug 24 22:18:05 2010 said:

Hi, I have attached a picture. I am aware that there is some EtBr that moves in the opposite direction of the DNA as seen in the last 9 wells. Thanks for your help Bob1 and others.

Which gel loading dye do you use?
Tried replace your running buffer (TBE, TAE etc?)

It looks like loading dye, otherwise the gel seems to be running fine.

Well,

I have recently changed the running buffer in the tank and it has made no difference.

We do not use a special gel loading dye before running the sample. Its PCR product straight into the gel. We use BIOTAQ Red DNA pol. for the PCR.

Hmmm, the "red" should migrate towards the positive electrode as well as the DNA, so I am not sure that it is the "red" after all. I guess it is still something from the PCR though. If you are trying to quantify the PCR product, you should clean it up before quantification as the dNTPs and fragments of DNA will affect the concentration. Also the "red" component may interfere with that reading, though it seems to be a proprietary substance, so it is hard to say for sure.

On second thought,
for the 9 wells far at the right, do you load anything or is just empty well? How thick is your gel and how deep is your comb?

It might due to ethidium bromide shift, although this will be less likely to happen. However such phenomenon "possibly" (i suspect) happen when you have a thin gel, but a deep comb where your comb almost touched the bottom of your molding plate, with only very little concentration of etbr. You also seems having a very long gel capture time (for UV, in mili seconds), or you possible left the bright light and UV light both turn on together in your gel-doc when you taking the picture.

bob1 on Thu Aug 26 23:33:44 2010 said:

Well Bob1,

My quantification attempts are independent of the PCR. PCR usually works well. We are trying to standardise this protocol and therefore quantification is important. So its surely nothing from the PCR product that might be interfering.

Thanks

adrian kohsf on Fri Aug 27 02:50:37 2010 said:

Adrian,

There is nothing loaded in the 9 wells at the right. So whatever, we see migrating upwards must be EtBr from the gel. But the places where the samples are loaded have darker spots.

I use a 2.5% agarose gel and the comb does not go very deep into the gel. Also, its just a UV transilluminator, so there is no inference from bright light.

Thanks for all your help people..... any suggestions on getting better OD after gDNA extraction are also welcome.

Ameya