Digoxigenin PCR Probe

dig-dUTP / dNTP Stock Mix:
dNTPs are individually purchased as 100 mM stocks from Pharmacia
    (#s: 27-2060-01, 27-2070-01, 27-2070-01, 27-2068-01)
dig-dUTP is purchased from Boehringer-Mannheim:
    Digoxigenin-11-dUTP (# 1 093 088) 25 nmol (25 ul)
    dNTPs and H20 are added to total dig stock to conc. below.

dATP		2.0 mM    3.04 ul (100 mM stock)
dCTP		2.o mM    3.04 ul (100 mM stock)
dGTP		2.0 mM    3.04 ul (100 mM stock)
dTTP		0.825 mM  1.26 ul (100 mM stock)
dig-dUTP 	0.165 mM  25.0 ul (as supplied)
                           117 ul H20
                       TOTAL VOLUME MIX: 152 ul

PCR in Cetus: 25 ul rxn

		H20  12.25	92C 3.5 min denature
(Promega M190A)	10X   2.5       92C 0.5 min 35 cycles
	(25mM)	Mg    1.5       55C 1.0 min
      dig-dNTP mix    2.5       72C 2.0 min
(5 pmol/ul)	P1    2.5       
   (5 pmol/ul)	P2    2.5
   (invisible)	DNA   1.0
(Promega 3U/ul)	Taq   0.25

Use directly with no further clean up.  Have used this reaction mix for in 
successful whole mount in situ hybridizations.

PCR in IDAHO: 25 ul rxn

		 H20  10.25	94C 60 sec denature
   (Idaho clear) 10X   2.5
	 (20 mM)  Mg   2.5	94C 15 sec
            dig-dNTP   2.5	55C 15 sec 35 cycles
      (5 pmol/ul) P1   2.5	74C 30 sec
      (5 pmol/ul) P2   2.5
(invisible)	 DNA   2.0	74C 60 sec
(Promega 3U/ul)	 TAQ   0.25

Use directly.  We used this scale reaction to screen about 12 large colony 
lifts plus 4 Southern blots.  Hyb was reused several times to accomplish this.
Used about 6-12 ul / 10 ml hyb solution.  Determine this emperically.  
Store probes frozen or in hyb mix.

Check incorporation:

1 ul + 5 ul SSC
92C 5 min
ice

2 ul > nitrocellulose
UV crosslink
wet w/2X ssc
wash twice PBST (5 ml / 5 min each)
10 min 5% NFDM in PBST
30 min 1:2000 antibody (1:8000 works)
4 washes PBST, 5 min each
Equilibration buffer
Stain per Boeringer protocol (phosphatase - NBT XPHOS)