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Promega Wizard Malfunction!

Promega Wizard Malfunction! - DNA Extraction Forum

Promega Wizard Malfunction! - DNA Isolation Forum. A forum specifically about questions on DNA extraction and purification.


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  #1  
Old 06-18-2013, 05:56 PM
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Angry Promega Wizard Malfunction!



Hi, I have a major problem during DNA extraction from whole blood using Promega Wizard, after the addition of nuclei lysis solution and protein precipitation and centrifuge, my supernatant remains bloody and after the Isopropanol step the pellet is not wholly white but rather turbid and kind of grayish
Please help!!!!!!
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  #2  
Old 06-19-2013, 03:37 PM
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Default Re: Promega Wizard Malfunction!

Despite the appearance of the pellet, does your DNA works for further processes (PCR, enzimatic digestion, cloning, etc)? I have never used the kit you mention but I have isolated DNA from blood before and I can tell you that a bloody supernatant is no good.

Still, I think you should try your DNA.

Regardless, I suggest you contact the support service of the company (the email ought to be printed on the kit's users manual or box) and ask about your problem.

Good luck
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Old 06-20-2013, 11:47 AM
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Default Re: Promega Wizard Malfunction!

Thank you for the reply, but can I ask you about the kit you use and there is something strange; I normally make duplicate of 300 um sample for three different patients and the two of the same person turn out bloody while the rest are clear. Does this give you more insight into my problem?
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Old 06-20-2013, 03:21 PM
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Default Re: Promega Wizard Malfunction!

If you are only having problems with the tubes of that patient then the problem is in the sample. Did you check its plasma, because it is posible that the blood sample is hemolysed. If what I say is true, the plasma should look red instead of yellow (the intensity of red shows the degree of hemolysis, the reder the worse).

Check that. If it is your case I suggest you lyse the red cells (erytrocytes) of the sample first and extract DNA from leucocytes instead from whole blood. You can do this for all the samples that gives you the problem you state (bloody supernatant and all that).

Hope this helps you. Good luck.
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  #5  
Old 06-21-2013, 07:48 AM
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Default Re: Promega Wizard Malfunction!

Thank you very much, I have contacted Promega live chat and their scientist told me to repeat lysing cells since during the original you have to lyse the RBCs in the first step.
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