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dna extraction from frozen blood

dna extraction from frozen blood - Basic Lab Protocols and Techniques

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  #1  
Old 06-25-2007, 03:44 PM
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Default dna extraction from frozen blood



hi
Can anyone tell me how to extract dna from blood(human) stored in a ordinary freezer.i have to separate serum first before going for the extraction but iam not able to.does anyone have a protocol for it.the samples are a month old.
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Old 07-27-2007, 01:49 AM
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Default Re: dna extraction from frozen blood

If you want to extract DNA from blood cells, you can thaw out your blood sample and pipette 0.5ml into new epi tube, add 0.5ml lysis buffer (tris, EDTA, SDS ph8) add Proteinase K, incubate 65C 1hour, boil 10min, centrifuge at max speed for 3 min, Run supernatant through Chromaspin Column or add 1vol phenol:chloroform:isoamyl alcohol, centrifuge 12,000g 10min, transfer upper aqueous layer to new tube, add 0.1Vol 3M NaAc and 2.5Vol 95% alcohol to precipitate. centrifuge at max speed 10min, remove sup, wash pellet 70%EtOH, centrifuge 10min 10000g, decant EtOH and dry pellet. Dissolve in ultrapure water. Treat w/ Rnase, if needed.
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  #3  
Old 07-27-2007, 02:05 AM
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Default Re: dna extraction from frozen blood

Quote:
Originally Posted by lakshb View Post
hi
Can anyone tell me how to extract dna from blood(human) stored in a ordinary freezer.i have to separate serum first before going for the extraction but iam not able to.does anyone have a protocol for it.the samples are a month old.
If you do not want to extract from the cells you can thaw out the blood sample, leave at RT to coagulate, centrifuge 3000g, remove supernatant, i.e. serum. Obviously if no coagulation takes place then your sample was treated with either citrate solution or EDTA solution or heparin (so now you will have plasma). In that case the 3000g centrifugation will still serve to remove whole cells. You can do extraction from the supernatant like I detailed in the post above.
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Old 07-28-2007, 03:53 PM
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Default Re: dna extraction from frozen blood

thanx man
for a wonderful reply
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Old 03-16-2009, 08:35 AM
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Default Re: dna extraction from frozen blood

can anyone tell me how to extract dna from blood(human)stored in a ordinary freezer.i have to separate blood mixed by serum.anyone have a protocol for it with salting out method.the samples are six month old.
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Old 03-19-2009, 03:41 PM
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Cool Re: dna extraction from frozen blood

If you do not want to extract from the cells you can:
thaw out the blood sample, leave at RT to coagulate, centrifuge 3000g, remove supernatant, i.e. serum. Obviously if no coagulation takes place then your sample was treated with either citrate solution or EDTA solution or heparin (so now you will have plasma).
In that case the 3000g centrifugation will still serve to remove whole cells.
Put 0.5ml into new epi tube, add 0.5ml lysis buffer (tris, EDTA, SDS ph8) add Proteinase K, incubate 65C 1hour,
boil 10min,
centrifuge at max speed for 3 min,
[Run supernatant through Chromaspin Column and you are done!]
or add 1vol phenol:chloroform:isoamyl alcohol, centrifuge 12,000g 10min, transfer upper aqueous layer to new tube, add 0.1Vol 3M NaAc and 2.5Vol 95% alcohol to precipitate. centrifuge at max speed 10min, remove sup, wash pellet 70%EtOH, centrifuge 10min 10000g, decant EtOH and dry pellet. Dissolve in ultrapure water.

Treat w/ Rnase, if needed.
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  #7  
Old 06-06-2011, 03:52 PM
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Default Re: dna extraction from frozen blood

Quote:
Originally Posted by danfive View Post
If you do not want to extract from the cells you can:
thaw out the blood sample, leave at RT to coagulate, centrifuge 3000g, remove supernatant, i.e. serum. Obviously if no coagulation takes place then your sample was treated with either citrate solution or EDTA solution or heparin (so now you will have plasma).
In that case the 3000g centrifugation will still serve to remove whole cells.
Put 0.5ml into new epi tube, add 0.5ml lysis buffer (tris, EDTA, SDS ph8) add Proteinase K, incubate 65C 1hour,
boil 10min,
centrifuge at max speed for 3 min,
[Run supernatant through Chromaspin Column and you are done!]
or add 1vol phenol:chloroform:isoamyl alcohol, centrifuge 12,000g 10min, transfer upper aqueous layer to new tube, add 0.1Vol 3M NaAc and 2.5Vol 95% alcohol to precipitate. centrifuge at max speed 10min, remove sup, wash pellet 70%EtOH, centrifuge 10min 10000g, decant EtOH and dry pellet. Dissolve in ultrapure water.

Treat w/ Rnase, if needed.
How do you thaw out the frozen blood.
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  #8  
Old 09-28-2011, 08:57 AM
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Default Re: dna extraction from frozen blood

What equipment do you use to extract the DNA?
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Old 01-11-2013, 08:05 PM
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