Peripheral Blood 96-well DNA Prep Protocol 

1.       Collect ~ 70 µl blood (by retro-orbital bleed) using a Heparanized capillary tube into 150 µl Saline EDTA in a 96-well plate (I like to use Rainin R96-OAPV-1CO v-bottom plates with Costar 3931 corner notch lids). 

2.      Spin plate at 2000 rpm for seven minutes in the Sorvall RT 6000B (or comparable) with swinging buckets.  (Note: This speed in this centrifuge with the appropriate plate carriers is very near the limit at which plates crack.  If you have problems with excessive plate cracking, I suggest experimenting with reducing the centrifuge speed by 100 RPM increments. The appropriate speed will likely still crack plate edges (mine does) but will not crack wells.)

3.       Remove supernatant by vacuum aspiration using a tabletop vacuum source with 8-channel head.

4.      Add 200 µl Buffone’s Lysis Buffer1 to wells and mix cells in buffer by pipetting up and down with a multi-channel pipetter.

5.      Spin plate at 2000 rpm for seven minutes in the Sorvall RT 6000B with swinging buckets (no hold time is required between addition of lysis buffer and commencement of spin step).

6.      Remove supernatant by vacuum aspiration using a tabletop vacuum source and 8-channel head.

7.      Repeat steps 4-6 for a second Buffone’s wash.

8.      Perform a third wash with Buffone’s (repeating steps 4-6) with the following changes:  Prior to the addition of the third Buffone’s wash, vortex the plate (after supernatant has been removed).  After Buffone’s is added, it will not be necessary to mix cells by pipetting.

9.      Vortex plate (after removal of supernadent).

10.  Resuspend the nuclear pellet (may not be detectable) in 100 µl Cell Lysis Buffer2 by pipetting several times and transfer to a 96-well PCR plate.

11.  Heat plate to 56oC for 1 hour in a thermocycler.

12.  Heat kill the Proteinase K by incubating at 97oC for 15 minutes in a thermocycler.

13.  Use 1 µl of the DNA solution in a 15 µl PCR.

 

1Buffone’s Lysis Buffer (500 ml)

0.32M Sucrose                         54.8 g Sucrose
10 mM Tris-HCl (pH7.5)            5 ml 1 M Tris-base
5 mM MgCl2                              2.5 ml 1 M MgCl2 

autoclave then add
1 % (v/v) Triton X-100             5 ml Triton X-100

 

2Cell Lysis Buffer (20 ml)

  18 ml Autoclaved ddH2O
   
    2 ml 10X AmpliTaq Gold Buffer (No MgCl2)
  20 µl Nonidet P40 (NP40)
  20 µl Tween-20
120 µl 10 mg/µl Proteinase K

 

It has been recommended to me that Cell Lysis Buffer be made fresh on day of use, but I have stored it up to two weeks at 4oC without notable degradation of quality.  It is also suggested to use autoclaved ddH2O which is not used for anything but PCR to avoid contamination.

 If any questions, contact Tom Sproule at tom.sproule@jax.org.