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DNA extraction impurities - (Nov/02/2013 )

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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 ?


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-sayk-

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.

-jerryshelly1-

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.

-Abdugaffar-

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?

-sayk-

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.

-El Crazy Xabi-

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.

-jerryshelly1-

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

-sayk-

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.

-jerryshelly1-

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?

-sayk-

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?

-jerryshelly1-
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