Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

GHOST in Electrophoresis


  • Please log in to reply
18 replies to this topic

#1 Ameya P

Ameya P

    Rervm Cognoscere Cavsas

  • Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 333 posts
26
Excellent

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
:)

NEW!!!!  Your hand wash can give you cancer  on CoffeeTableScience!!!! 

Image copyright: Adrian Koh SF.
Replication of this art is strictly prohibited without express permission of the artist


#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,830 posts
414
Excellent

Posted 24 August 2010 - 02:18 PM

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

#3 Ameya P

Ameya P

    Rervm Cognoscere Cavsas

  • Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 333 posts
26
Excellent

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.
:)

Attached Files


NEW!!!!  Your hand wash can give you cancer  on CoffeeTableScience!!!! 

Image copyright: Adrian Koh SF.
Replication of this art is strictly prohibited without express permission of the artist


#4 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 708 posts
28
Excellent

Posted 25 August 2010 - 08:15 AM

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

Edited by adrian kohsf, 25 August 2010 - 08:17 AM.

Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#5 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,830 posts
414
Excellent

Posted 25 August 2010 - 04:28 PM

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

#6 Ameya P

Ameya P

    Rervm Cognoscere Cavsas

  • Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 333 posts
26
Excellent

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.

NEW!!!!  Your hand wash can give you cancer  on CoffeeTableScience!!!! 

Image copyright: Adrian Koh SF.
Replication of this art is strictly prohibited without express permission of the artist


#7 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,830 posts
414
Excellent

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.

#8 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 708 posts
28
Excellent

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.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#9 Ameya P

Ameya P

    Rervm Cognoscere Cavsas

  • Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 333 posts
26
Excellent

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 :)

NEW!!!!  Your hand wash can give you cancer  on CoffeeTableScience!!!! 

Image copyright: Adrian Koh SF.
Replication of this art is strictly prohibited without express permission of the artist


#10 Ameya P

Ameya P

    Rervm Cognoscere Cavsas

  • Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 333 posts
26
Excellent

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

NEW!!!!  Your hand wash can give you cancer  on CoffeeTableScience!!!! 

Image copyright: Adrian Koh SF.
Replication of this art is strictly prohibited without express permission of the artist


#11 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,830 posts
414
Excellent

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.

#12 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
16
Good

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.

#13 Ameya P

Ameya P

    Rervm Cognoscere Cavsas

  • Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 333 posts
26
Excellent

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

NEW!!!!  Your hand wash can give you cancer  on CoffeeTableScience!!!! 

Image copyright: Adrian Koh SF.
Replication of this art is strictly prohibited without express permission of the artist


#14 Ameya P

Ameya P

    Rervm Cognoscere Cavsas

  • Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 333 posts
26
Excellent

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 :)

NEW!!!!  Your hand wash can give you cancer  on CoffeeTableScience!!!! 

Image copyright: Adrian Koh SF.
Replication of this art is strictly prohibited without express permission of the artist


#15 donny

donny

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 74 posts
3
Neutral

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.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.