dna extraction from frozen blood

[lakshb]

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.

[danfive]

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.

[danfive]

Originally Posted by lakshb

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.

[lakshb]

thanx man
for a wonderful reply

[gmobini]

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.

[danfive]

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.