DNA from plasma

Friday, October 24, 2003

Description
Sometimes the only material available on a subject is stored plasma or serum samples. The DNA content in such samples is generally low, but sufficient to serve as template for PCR reactions. Many disease states will increase plasma DNA concentrations, and small amounts of fetal DNA have been detected in maternal plasma/serum during gestation.

2.

3. DNA Extraction:
3.1

Procedure
1. Place 1.5ml serum or plasma into a 15ml centrifuge tube.
2. Add 1.5 ml of 1X SDS proteinase K solution in the tube containing the serum and mix well.
3. Digest overnight at 55 C in water bath.
4. Add 3 ml Phenol/chloroform solution.
5. Vortex 30 seconds and centrifuge for 10 min at 2500 rpm. using a swing-out rotor.
6. Transfer aqueous layer to fresh tube and repeat steps 4-5.
7 Transfer aqueous layer to fresh tube and add 5 ul glycogen (10 mg/ml) 1 ml 7.5 M Ammonium acetate, and 8 ml 100% ethanol .
8. Mix by inverting and centrifuge the at 6000 rpm for 40 minutes.
9. Carefully remove supernatant and wash pellet in 10 ml 70% ethanol.
10. Centrifuge at 6000 rpm for 10 minutes. Carefully remove last traces of ethanol, and allow to air dry for 10 min before re-dissolving in 100 ml TE

Recipes
10X SDS /Protein K:
Lauryl sulfate (SDS) 10 %
Proteinase K 5 mg /ml.
TE
Phenol/chloroform

Supplies

Tips

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DNA from plasma