Procedure for Microplate DNA Hybridization Assay

1) Perform PCR reaction.

2) Bring water bath to temp (60°C) preferably the night before.

3) Preheat Hybridization Solution, High Stringency Buffer, and Conjugate Dilutant to 37 ºC.

4) Bring plates to room temperature

5) Place 10 ul of PCR reaction into each well.

6) Add 5ul of Denaturation Solution (0.25N NaOH) to each well.

7) Incubate at room temp. for 2 mins.

8) ADD 85 ul of Hybridization Solution (0.43 M phosphate buffer, pH 6.8, 0.1% SDS, 5X Denhardt’s) to each well.

9) Seal and Incubate in 60ºC water bath for 30 minutes.

10) Check (rinse) plate washer.
   TK1 program, indicate number of strips
   Run with water to check (make sure water is connected in back) It will wash three times, make sure all spouts are working properly.

11) Wash plate 3 times at room temp with 1X Wash Solution (using plate washer).

12) ADD 200 ul High Stringency Buffer to each well.

13) Seal and Incubate in a 60ºC water bath for 20 min.

14) Wash plate 3 times at room temp with 1X Wash Solution (using plate washer).
NOTE: Steps 12-18 are extremely time sensitive. For best results, make sure steps 12-18 steps are carried out efficiently.

15) Dilute the Streptavidin-POD Conjugate (Roche Applied Science #1089153) 1:5000 in Conjugate Dilutant.
NOTE: for 1 plate, ADD 2 ul Streptavidin-POD to 10 ml Conjugate Dilutant. Throw out extra. (The Streptavidin-POD arrives as 500 units of powder. Reconstitute in 1 ml autoclaved MQ. This yields 0.5 units/ul. So 2 ul/plate = 1 unit plate. Seal vial tightly to prevent drying).

16) Add 100ul diluted conjugate to each well.

17) Seal plate and incubate at 37 ºC for 10 minutes.

18) Wash 3 times at room temperature with 1X Wash Solution (using plate washer).

19) ADD 100 ul of TMB 1 Component HRP Microwell Substrate (BioFX Laboratories #TMBW010004) (TMB = Tetramethyl Benzidine) to each well. Measure amount needed before pouring in basin, chemical is expensive.

20) Seal and incubate at 37 ºC for 5-10 minutes.

21) ADD 100 ul Stop Solution (0.5M HCL) to each well.

22) Measure absorbance at 450 nm on plate reader within 10 minutes.

23) Rinse line of plate washer.

PROCEDURE for T-tailing probe:
Our Method for 25 plates:
1) Combine in a labeled 15 ml conical tube:
   50 ul 5x Tdt buffer = 1.2X Tdt buffer (Roche Molecular Diag.)
   100 ul autoclaved MQ water
   10 ul 100 mM dTTP = 4.8 mM dTTP (Roche Molecular Diag.)
   25 ul 100 pmol/ul oligo = 2.5 nmol oligo
   10 ul x 400 units/ul = 4000 units of Tdt enzyme; (Roche Molecular Diagnostics #3333566)
   5 ul 10 mg/ml inorganic pyrophosphatse (Sigma) = 0.25 mg/ml
   200 ul total volume

2) Incubate in 37ºC water bath for 4 hours.

3) Remove from water bath, Add 42.5 ul of 0.5 M sterile EDTA, pH 8.0.

4) Add 2.25 ml 1X sterile TE, pH 7.4.
NOTE: this gives T-tailed probe at a final concentration of 1 pmol/ul

5) Store T-tailed probe at ­20 ºC.

PROCEDURE for Probing A Plate:
24) Defrost T-tailed probe. (see below on how to T-tail)

25) Take 100 ul of T-tailed probe, ADD 10 ml sterile 1X TE (pH 7.4).

26) ADD 100 ul of this mixture into each well (this uses 9.6 ml per plate).
NOTE: this gives 1 pmol of T-tailed probe per well.

27) Dry in oven (37-50ºC) overnight or until dry.

28) ADD 200 ul/well of Blocking Buffer (5X Denhardt’s Solution, 1X phosphate buffered saline (PBS)).

29) Incubate at room temp 1-4 hr.

30) Dump buffer in sink, and shake out excess on absorbent paper towels.

31) Dry upside down at room temp for 1-2 hrs or until completely dry.

32) Seal plates with plate sealer and store in sealed bag with desiccant at 4ºC.

References:
T. Kiesling, M. Diaz, A. Statzell-Tallman, and J.W. Fell (2002) Field identification of marine yeasts using DNA hybridization macroarrays. In: Marine Mycology: The Organisms, Ecology and Applied Aspects. Hyde, K. (ed). Hong Kong: Fungal Diversity Press, 69-80.

K.D. Goodwin, S.A. Cotton, G. Scorzetti, and J.W. Fell (2005) A DNA hybridization assay to identify toxic dinoflagellates in coastal waters: detection of Karenia Brevis in the Rookery Bay National Estuarine Research Reserve. Harmful Algae, 4:411-422