4,931,223 12
added and allowed to soak in for approximately one -continued
minute.
The assay membrane is placed in a camera luminome- Technical Data Shm.
2 ml 20 X SSFE
ter device equipped with pre-exposed calibration scales 4 ml 20% SUS
for P-HCG and LH. 5 394 ml ddh2O
The chemilminescent fight emission generated as a 400 in] (heated)
function of the craymes, alkaline phosphatane and car- Wash Buffer III
boxyl esterase, is imaged through a mask containing 0.1 x SSPE/0.1% SDS
two narrow, band pass filters (approximately I cut in 2 .0 20 x SSPE
dia ter). Kodak Wmttcn Gelatin Filter No. 115 is 10 4 ail 10% SDS
used to urage green emission from the benzDpyranyl 394 in[ Milo
1,2-diametame substrate, and Kodak Wratten Filter No. 400 ml
47B is used ' to isolate the blue emission from the phenyl Wash Baser tV
diosetane. 3 mM Tris-RCI (pH 9.5)
The relative levels ofR-HCG and HIE present in the 15 0.6 ml I M Tritina Rate
simple are determined by a comparison ofthe appropni- L199.4 ad ddH20
ate imaged spot brightness with relevant calibration 200.0 ad
scales. Wash Buffer V
EXAMPLE 11 20 0.1 M Tricaut HCI pH 6.0
101) X Dcalum's actinic.
A three-chamml analysis for Herpes Simplex Virus,
(HSV), Cytomegdovirm, (CMV), and Human
Papiloma Vinut, (HFV) is carried out as follows: Preparation of 100 X Denhurs solution (for 100 nds)
MATERIALS 7.5 Polyvinylpyrrolidom (2 g; until. wt. 40 K, PVP-40)
and 2 g firoll are dissolved at a temperature greater than
"Gene Screen Plus", a positively charged nylon 65' C. but less than boiling. The solution is cooled to
membrane (Dupont NEN Products) is used for hybridi. approximately 44Y C, 2 g BSA are added and the solu,
zation. tion is brought up to the final volume of 100 in] with
The following buffers arc used for the assay- 30 ddRzO, aliquoted and stored at -20' C BSA is easily
HSV DNA PROBE ASSAY denatured Bad in combination with PVP and ficoll it
will not go into solution at all if it is not cooled down to
Materials and Buffers -4W C. Hence, the solution is not heated over 40* C.
Membrane: Octic Screen Plus membrane. when thawing for use.
Bufferse Denaturation Buffer, 0.5 M NaOH 35
Neutraliauttion Buffer, 0.4 M NaH2PO4 (pH=2.0) Preparation of 20 X SSC solution
Loading Buffer, I part Denaturation Buffer I part Neu- 20 x SSC (for too ad)
tralization Buffer 3.0 M Sodium Chloeid. 17.4 g
Membrane Wetting Buffer 0.4 M Tris (pH=7.5) 03 M Sodium ritime 9.9 a
40
Membrane, Prehytindization Buff" The volume is brought to 100 ml and the solution
S Final Concentration filtered through a 0.45 pat nitrocellulose filter and
0.5 ad 100 x Denhardt's 5 X stored at room temperature.
solution 45 Preparation of 20 X SSPE solution
0.5 nd 10% SDS 0.5%
2 5 ML 2D x SSPE 5 X 20 X SSFE PH 7.4 (for I liter)
2n mg denatured, 20D lg/nd 3.6 M NaCT
samicated salintim
spare DNA 200 mM Sodium phosphate 23 g dibasic, 5.92g monoba
ddH20 sic
10 ad 50 20 mM EDTA 7." g
Membiatic Hylinduatum Bdlu These materials are dissolved, adjusted to PH 7.4 with
Final Concentration 5N NaOH, brought to a volume of I liter and the solu
tion is then filtered through a 0.45 ý= nitrocellulose
0.5 rid IOD x Dethardt's solution 5 X
0.5 ad 10% SDS 0.5% filter.
2.5 ml 20 x SSPE 5 X 55
2.0 ing salmon spenin DNA 200 tiglail
2.0 ml 50% Daman sullatie IN I X TE
ý dk[E[20 I x TE buffe, 10 mM TrL (pH 7.)
10 ml I mM EDTA
Wash Buffff 1 60 Autoclave
I X SSPE/0.1% SDS
20 ml 20 X SSPE The substrate buffer solution contains 0.05M carbon
4 ml 10% SDS ate6 I mM MgCl2, 0.1% by weight BSA (Ph=9.5) and
376 in] ddH20 3-(2'-spiroadý ntme)4.methoxy-4-(3-amtoxy)phe
400 mi 65 nyl-1,2-dioxetane disodium salt (50 mg/ml), 3-(2'
Wash Buffer 11 spiroadamantaLne)4-(7"-phosphoryloxy-4"-trifluorome
0.1 X SSPE/0.1% SDS peaheated thyl)beaw-2H-pyran-2'-yl-1.2-dic;xetane, (50 mg/ml)
to Wait, tamperatiare indicated and 3-(2-spirgadmmtme)-4-(3"-bomothinol-2-yl-7"-
Page: Previous 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Next
Last modified: November 3, 2007