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
- - - - -

DNA extraction impurities

dna extraction promega impurities

  • Please log in to reply
23 replies to this topic

#1 sayk

sayk

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 02 November 2013 - 10:14 AM

I use promega wizard DNA extraction kit

 

I usually make a duplicate of each of my samples, the step where we add nuclei lysis solution and protein precipitation solution then centrifugation the dark brown pellet is evident but the solution is red and this sometimes occurs to one of the duplicate not both, one being clear while the other red or reddish and after transferring the solution to isopropanol tubes and centrifugation and alcohol addition and centrifugation the pellet looks turbid and sometimes brownish.

 

I attached some pictures of my results

 

Help what should I do ?

Attached Thumbnails

  • after alcohol and centrifugation(duplicates of same sample).jpg
  • after isopropanol and centrifugation.jpg
  • after nuclei lysis soln and protein ppt soln 2.jpg


#2 jerryshelly1

jerryshelly1

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 328 posts
29
Excellent

Posted 02 November 2013 - 01:50 PM

If you are extracting blood, it looks like your protein precipitation buffer is no longer viable. The red color is due to the protein hemoglobin being present in the solution. I would double check your protocol and make sure that you are allowing for proper precipitation of your protein and also check to see if your precipitation buffer is coming out of solution.

 

Are you centrifuging your samples adequately each time? Is it possible you are overloading the kit with blood each time?

 

As far as the brown pellet, it looks like you still have cellular debris in your sample.



#3 Abdugaffar

Abdugaffar

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 02 November 2013 - 11:09 PM

I am using QIAamp DNA mini Kit in order to extract DNA from buccal swab.

 

I wonder the difference between Buffer ATL (tissue lysis and) and Buffer AL (cell lysis). And also, Buffer AW1 and Buffer AW2 during washing step.

Those questions taking place in my mind what difference and what kind of ingredients they contain.



#4 sayk

sayk

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 03 November 2013 - 09:00 AM

If you are extracting blood, it looks like your protein precipitation buffer is no longer viable. The red color is due to the protein hemoglobin being present in the solution. I would double check your protocol and make sure that you are allowing for proper precipitation of your protein and also check to see if your precipitation buffer is coming out of solution.

 

Are you centrifuging your samples adequately each time? Is it possible you are overloading the kit with blood each time?

 

As far as the brown pellet, it looks like you still have cellular debris in your sample.

thank you but It's a brand new kit, when I faced this problem I ordered a new one, and I follow the protocol precisly,

other suggestions?



#5 El Crazy Xabi

El Crazy Xabi

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 192 posts
20
Excellent

Posted 03 November 2013 - 04:22 PM

I am using QIAamp DNA mini Kit in order to extract DNA from buccal swab.
 
I wonder the difference between Buffer ATL (tissue lysis and) and Buffer AL (cell lysis). And also, Buffer AW1 and Buffer AW2 during washing step.
Those questions taking place in my mind what difference and what kind of ingredients they contain.


According to the MSDS and QIAGEN's web, ATL is a buffer containing SDS to help in the lysis when incubated with proteinase K, while the AL is a guanidinium chloride-based buffer needed to bind the DNA to the silica membrane.


Edited by El Crazy Xabi, 03 November 2013 - 04:23 PM.


#6 jerryshelly1

jerryshelly1

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 328 posts
29
Excellent

Posted 04 November 2013 - 08:28 AM

Are you using fresh blood and/or are you properly resuspending it before beginning the isolation? If it is older blood, it may contain fibrin which can induce clotting and will make it more difficult to adequately remove all protein.



#7 sayk

sayk

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 05 November 2013 - 08:45 AM

Are you using fresh blood and/or are you properly resuspending it before beginning the isolation? If it is older blood, it may contain fibrin which can induce clotting and will make it more difficult to adequately remove all protein.

 

it is a day old blood, what do you mean resuspending it before beggining the isolation?

thank you in advance



#8 jerryshelly1

jerryshelly1

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 328 posts
29
Excellent

Posted 05 November 2013 - 08:48 AM

It was a silly question, but I am just making sure the blood was homogenous before you began your prep. Blood will separate into its respective components fairly quickly and I have seen people take the plasma or the RBC's while leaving the buffy coat almost undisturbed.



#9 sayk

sayk

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 05 November 2013 - 09:49 AM

It was a silly question, but I am just making sure the blood was homogenous before you began your prep. Blood will separate into its respective components fairly quickly and I have seen people take the plasma or the RBC's while leaving the buffy coat almost undisturbed.

 

how can I make sure that I take mostly the buffy coat?

do you think the problem I'm facing has anything to do that the pateints have cancer and take chemo therapy?



#10 jerryshelly1

jerryshelly1

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 328 posts
29
Excellent

Posted 05 November 2013 - 10:31 AM

It may affect your DNA concentration, but not the residual protein. If they are immune compromised (i.e. low WBC's) you would expect a lower than average DNA concentration, but the overall consistency of the blood would be similar.

 

As far as the chemotherapy, I do not have enough knowledge on that to make a guess. Are you only seeing this phenomenon in you cancer and chemo patients? Do your healthy controls show this?

 

I checked the wizard protocol and it is very straight forward. Do you see any clumps in the whole blood before you begin your extraction? Is this a recent problem that was absent in the past?



#11 sayk

sayk

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 05 November 2013 - 10:56 AM

It may affect your DNA concentration, but not the residual protein. If they are immune compromised (i.e. low WBC's) you would expect a lower than average DNA concentration, but the overall consistency of the blood would be similar.

 

As far as the chemotherapy, I do not have enough knowledge on that to make a guess. Are you only seeing this phenomenon in you cancer and chemo patients? Do your healthy controls show this?

 

I checked the wizard protocol and it is very straight forward. Do you see any clumps in the whole blood before you begin your extraction? Is this a recent problem that was absent in the past?

 

Thanks for your thorough reply.

 

I've faced this problem since the beginning with both cases and controls, there were no clumps in the blood and the same problem happened when I extracted the DNA from

 

samples drawn on the same day by professionals. 



#12 jerryshelly1

jerryshelly1

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 328 posts
29
Excellent

Posted 05 November 2013 - 11:44 AM

The only thing I can suggest is taking some "junk" blood and running a couple of different conditions side by side.

 

Maybe...

Increase centrifugation time on a sample, precipitate protein at 4C for 10min?, increase amount of precipitation solution?



#13 sayk

sayk

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 05 November 2013 - 12:30 PM

The only thing I can suggest is taking some "junk" blood and running a couple of different conditions side by side.

 

Maybe...

Increase centrifugation time on a sample, precipitate protein at 4C for 10min?, increase amount of precipitation solution?

 

I've tried all that except precipitation at 4C for 10, I'll try it tomorrow

thanks



#14 hercolanium

hercolanium

    member

  • Active Members
  • Pip
  • 14 posts
0
Neutral

Posted 14 November 2013 - 03:01 AM

Hi, I am having the same problem so I have tried your instructions by incubating the samples at 4 C with the Nuclei lysis solution and Protein Precipitation solution, the 1-2 days old samples came out bloody and impure, the DNA pellet came out brown like it has been burned after transfering to isopropanol tube and centrifugation

Please help blink.png



#15 jerryshelly1

jerryshelly1

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 328 posts
29
Excellent

Posted 14 November 2013 - 07:20 AM

PM Sayk and see if he/she worked out the problem.







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

©1999-2013 Protocol Online, All rights reserved.