DNA isolation from blood

A1542 MSDS 乙酸铵

for molecular biology, ≥98% (Sigma)

A1542-500G 340.47

销售说给九五折，明天处理

DNA isolation from blood

Solutions needed:

155 mM NH4Cl 8.293g

10 mM KHCO3 1g

0.25 mM EDTA (optional preservative) 0.084

50 mM Tris-HCl, ~pH 8.0 6.055

10 mM EDTA 0.34

1% SDS

7.5 M Ammonium Acetate（乙酸胺） 585G

100% 2- propanol（异丙醇）

70% ethanol（乙醇）

ddH20

we made some modification on:

> 1 separate and store the serum

> 2 normalize RBC lysis buffer + plasma to 45 ml per tube (~13 ml blood+32 RBC) > 3 normalize WBC lysis buffer to 7ml to resuspension from step above

> 4 adding 2.5 ml Protein Precipitation per tube

> 5 centrifuge 8000 rpm x 15 min @ step 10 & 13

> 6 centrifuge 8000 rpm x 10 min @ step 16

We use the Puregene (from Gentra, now Qiagen) protocol with 2 modifications:

It can be easily scaled to whatever needed volume. See attached protocol booklet.

Protocol: DNA Purification from Whole Blood or Bone

Marrow Using the Gentra Puregene Blood Kit

This protocol is for purification of genomic DNA from fresh or frozen samples of 5 ml whole blood using the Gentra Puregene Blood Kit. The protocol can also

be used for DNA purification from packed cells, buffy coat, or bone marrow. Important points before starting

■ Bone marrow samples may contain more white blood cells than a whole blood sample. After addition of Cell Lysis Solution, make sure that the solution is

homogeneous. If the solution is not homogenous, add additional Cell Lysis Solution and scale the volumes of the other reagents used in the protocol accordingly. ■ Frozen blood and bone marrow samples should be thawed quickly in a 37°C water bath with mild agitation and stored on ice before beginning the procedure. Things to do before starting

■ Preheat water bath to 65°C for use step 19 of the procedure.

■ Optional: Preheat water bath to 37°C for use in step 8 of the procedure.

Procedure

separate and store the serum

1. Dispense 10ml RBC Lysis Solution into a 15 ml centrifuge tube.

2. Add 5ml whole blood or bone marrow, and mix by inverting 10 times.

3. Incubate 10 min at room temperature (15–25°C). Invert at least once during the incubation.

4. Centrifuge for 5 min at 2000 x g to pellet the white blood cells.(twice)

5. Carefully discard the supernatant by pipetting or pouring, leaving approximately 200 μl of the residual liquid and the white blood cell pellet.

6. Vortex the tube vigorously to resuspend the pellet in the residual liquid. Vortexing greatly facilitates cell lysis in the next step.

The pellet should be completely dispersed after vortexing.

7. Add 5 ml Cell Lysis Solution, and pipet up and down to lyse the cells or vortex vigorously for 10 s.

Usually no incubation is required; however, if cell clumps are visible, incubate at 37°C until the solution is homogeneous.

Samples are stable in Cell Lysis Solution for at least 2 years at room temperature.

8. Optional: If RNA-free DNA is required, add 25 μl RNase A Solution, and mix by inverting 25 times. Incubate for 15 min at 37°C. Then incubate for 3 min on ice to quickly cool the sample.

9. Add 2.5 ml Protein Precipitation Solution, and vortex vigorously for 20 s at high speed.

10. Centrifuge for 8000 rpm x 15 min.

The precipitated proteins should form a tight, dark brown pellet. If the protein pellet is not tight, incubate on ice for 5 min and repeat the centrifugation.

11. Pipet 5 ml isopropanol into a clean 15 ml tube and add the supernatant from the previous step by pouring carefully.

Be sure the protein pellet is not dislodged during pouring.

12. Mix by inverting gently 50 times until the DNA is visible as threads or a clump.

13. Centrifuge for 8000 rpm x 15 min

The DNA may be visible as a small white pellet.

14. Carefully discard the supernatant, and drain the tube by inverting on a clean piece of absorbent paper, taking care that the pellet remains in the tube.

15. Add 5 ml of 70% ethanol and invert several times to wash the DNA pellet.

16. Centrifuge for 1 min at 8000 rpm x 10 min

17. Carefully discard the supernatant. Drain the tube on a clean piece of absorbentpaper, taking care that the pellet remains in the tube. Air dry the pellet for 1 min. The pellet might be loose and easily dislodged.

Avoid over-drying the DNA pellet, as the DNA will be difficult to dissolve.

18. Add 500 μl DNA Hydration Solution and vortex for 5 s at medium speed to mix.

19. Incubate at 65°C for 1 h to dissolve the DNA.

20. Incubate at room temperature overnight with gentle shaking. Ensure tube cap is tightly closed to avoid leakage. Samples can then be centrifuged briefly and transferred to a storage tube.