From Whole Blood for Pcr

FROM WHOLE BLOOD FOR PCR

1. Obtain 65ul-100ul of blood by retroorbital bleed with a microcapillary tube. Expel blood immediatly into 1ml Saline:EDTA in a 1.5 ml microfuge tube and MIX VIGOROUSLY!!! Do not allow any clot formation !!!!!!! Store on ice until processing.

2. Centrifuge 2000 rpm for 7 min. @ 4°c in a table-top centrifuge (or a variable speed microfuge ie: Eppendorf @ 3.5 ~= 850-900 x g.).

3. Begin to thaw out PBND, make Pro K solution, and heat water bath to 55°c.

4. Pipet off supernate . AVOID ASPIRATING THE LYMPHOCYTES !!!!!!!!!

5. Add 100ul Buffones Lysis Buffer to each tube and vortex. Break up any clumps that occur.

6. Microfuge on "HI" (setting#14) 25 secs.

7. Remove supernate and repeat steps #4 (with 200ul Lysis buffer) , #5 and #6 two more times. MAKE CERTAIN NO RED/PINK COLOR PERSISTS AS THIS WILL INHIBIT PCR.

8. Resuspend lymphocyte pellet in 100ul PBNDP (PBND with Pro K) and incubate @ 55°c for 60 min.

9. Heat samples to 97°c for 10 min in PCR machine (or by boiling).

10. Add 1-5ul* processed blood (DNA) / 25ul PCR reaction. * this volume will vary depending on your particular PCR primers, their Tm, and the level of nucleated cells in the periphery .

REAGENTS:

1) Buffones Lysis Buffer 500 ml

0.32 M Sucrose 54.8 g sucrose

10mM Tris-HCl (pH 7.5) 5 ml 1M stock Tris-HCl

5 mM MgCl2-6H2O 508 mg MgCl2-6H2O

1% v/v Triton X-100 5 ml Triton X-100

2) (PBND)PCR Buffer w Nonionic Detergents 500 ml

50 mM KCl 1.87 g KCl

10 mM Tris-HCl (pH 8.3) 5 ml 1M Tris-HCl Stock

2.5 mM MgCl2- 6H2O 255 mg MgCl2- 6H2O

0.1 mg/ml gelatin 50 mg gelatin

0.45% v/v Nonidet P40 (NP40) 2.25 ml NP40

0.45% v/v Tween 20 2.25 ml Tween 20

q to 500 ml w ddiH2O and autoclave

Store frozen in 5 ml aliquots

3) Proteinase K : Made fresh for each use !

10 mg Proteinase K is dissolved in 1 ml ddiH2O @ 4°c.