GHOST in Electrophoresis (View forum version)



Ameya P

Posted 24 August 2010 - 05:30 AM

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
:)

bob1

Posted 24 August 2010 - 02:18 PM

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

Ameya P

Posted 24 August 2010 - 08:48 PM

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


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.
:)


Adrian K

Posted 25 August 2010 - 08:15 AM

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

bob1

Posted 25 August 2010 - 04:28 PM

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

Ameya P

Posted 25 August 2010 - 08:33 PM

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.

bob1

Posted 26 August 2010 - 03:33 PM

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.

Adrian K

Posted 26 August 2010 - 06:50 PM

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.

Ameya P

Posted 26 August 2010 - 08:57 PM

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.


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 :)

Ameya P

Posted 26 August 2010 - 09:04 PM

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.


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

bob1

Posted 29 August 2010 - 01:37 PM

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

HomeBrew

Posted 29 August 2010 - 07:12 PM

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.

Ameya P

Posted 29 August 2010 - 09:53 PM

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

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

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.

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

Ameya

Ameya P

Posted 17 September 2010 - 11:40 PM

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


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 :)

donny

Posted 18 September 2010 - 02:38 AM

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.

Ameya P

Posted 19 September 2010 - 08:42 PM

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.


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.

HomeBrew

Posted 20 September 2010 - 04:07 AM

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.

Ameya P

Posted 20 September 2010 - 04:56 AM

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.


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.

HomeBrew

Posted 20 September 2010 - 06:20 AM

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.