Revised version - December 2, 1997

Covalent Binding of Solid Phase Primer

1. Make sure that your solid phase primer is phosphorylated or aminated at the 5'-end and that a linker of at least 10 T's (thymidine) is added to the 5'-end of the primer.

2. Prepare a freshly made coating mix consisting of 100 nM solid phase primer and 10 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) (Sigma, Cat. No. E 7750) in 10 mM 1-methyl-imidazole (1-MeIm) (pH 7.0).

3. Add 100 µl of this coating mix to each well. This gives a total of 10 pmol added to each well of the 5´-phosphorylated solid phase primer.

4. Seal the NucleoLink Strips (e.g. with Nunc Sealing Tape, Cat. No. 236366).

5. Incubate the NucleoLink Strips at 50°C for 4-24 hours.

6. To remove coating solution residues, wash the empty NucleoLink wells three times, soak for 5 min and wash three times, all with 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20 at room temperature (RT). Empty the Strips.

7. To remove salt residues, wash once with deionized sterile water.

8. The empty, coated NucleoLink Strips can be stored at 4°C or below in an ethylene bag. The Strips should not be sealed.

Amplification

1. BSA must be added to the PCR mix in a final concentration of 1 mg/ml. This concentration should not be exceeded as this may block the solid phase amplification. Furthermore, a concentration of 0.1%-0.25% Tween 20 is recommended.

2. Both primers must be added to the PCR mix. The concentration of the two primers should be in a ratio of 1:8 with the primer used as the solid phase primer in the lowest concentration. At Nunc A/S Research Laboratory the concentration used is 25 pmol per reaction of the primer not used as the solid phase primer and 25/8 pmol per reaction of the primer used as the solid phase primer.

3. Add PCR mix to the wells (normally 20 µl or 45 µl).

4. Add DNA template to each well (the total reaction volume has been tested with both 25 µl and 50 µl).

5. Seal the NucleoLink Strips with Tape 8 (Nunc Cat. No. 249719).

6. Place the Strips in a thermal cycler block.

7. Place the silicone spacer plate (Nunc Cat. No. 250150) on the tape sealed NucleoLink Strips and tighten the heated lid firmly.

8. Temperature cycle the Strips with the temperatures and cycling parameters specific for the system.

9. Remove the NucleoLink Strips and empty them. For further analysis of the liquid phase, it can be stored in GeNunc™ wells (GeNunc™ 120, Cat. No. 232549) at 4°C in a sealed ethylene bag.

10. Wash the empty NucleoLink wells three times, soak for 5 min, and wash three times, all with freshly made 0.2 M NaOH and 0.1% Tween 20 at RT.Wash the empty NucleoLink wells three times, soak for 5 min, and wash three times, all with 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20 at RT.

Detection

1. Add 100 µl of 50-100 nM biotinylated hybridization probe diluted in 5 ´ SSC, 0.1% Tween 20, and 0.5% blocking reagent (BR) (Cat. No. 1096176. Boehringer Mannheim, Germany) to each well.

2. Seal the NucleoLink Strips with Nunc Sealing Tape and incubate at 45-50°C for one to 20 hours (probe dependent).

3. Wash the empty NucleoLink wells three times at RT with 0.5 ´ SSC and 0.1% Tween 20.

4. Soak for 15 min at 50°C with 0.5 ´ SSC and 0.1% Tween 20.

5. Wash three times at RT with 0.5 ´ SSC and 0.1% Tween 20.

6. Detection of biotin-label on the hybridized probe:

1. When using alkaline phosphatase (AP): Add to each well 100 µl AP conjugated streptavidin (code No. D0396, DAKO, Denmark) diluted 1:3000 (or as the producer suggests) in 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20, and 0.5% BR.

2. When using Horse Radish Peroxidase (HRP): Add to each well 100 µl HRP conjugated streptavidin (Code no. P-0397. DAKO, Glostrup, Denmark) diluted 1:5000 (or as the producer suggests) in 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20, and 0.5% BR.

7. Incubate for one hour at 37°C - 50°C sealed with Nunc Sealing Tape.

8. Wash the empty NucleoLink wells three times, soak for 5 min, and wash three times with 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20 at RT.

9. Two substrates have been tested with AP: 4-MUP (4-methylumbelliferyl phosphate) and pNPP (para nitrophenylene phosphate). One colour forming reagent has been tested with HRP, i.e. TMB (3,3',5,5'-tetramethylbenzidine) in a ready-to-use solution (Cat. No. 4380H. Kem-En-Tec, Denmark).

1. When using 4-MUP: Add 100 µl of 1 mM 4-MUP dissolved in 1 M diethanolamine (pH 9.8) and 1 mM MgCl₂ to each well.

2. When using pNPP: Add 100 µl of 1 or 10 mg/ml pNPP in 1 M diethanolamine (pH 9.8) and 1 mM MgCl₂ to each well.

3. When using TMB: Add 100 µl of the ready-to-use solution to each well.

10. Substrate incubation.

The NucleoLink Strips should be sealed with Nunc Sealing Tape when incubated for more than 30 min

1. When using 4-MUP, incubate at 37-50°C in the dark for 30-60 min Add 50 µl of 3 M K₂HPO₄, to stop the hydrolyzation of 4-MUP.

2. When using pNPP, incubate at RT for 30 min (10 mg/ml) to 24 hours (1 mg/ml). Add 100 µl of 1 M NaOH to stop hydrolyzation of pNPP.

3. When using TMB, incubate for 30 minutes at RT. Add 100 µl 0.1 M H₂SO₄ to stop the reaction. Note: Rehybridization is not possible after addition of acid.

11. To measure the signal.

1. For detection of hydrolyzed 4-MUP, determine the signal in a fluorescence plate reader: Excitation wavelength 360 nm, emission wavelength 450 nm (also if the reaction has been stopped with K₂HPO₄).

2. For detection of hydrolyzed pNPP, measure OD in a normal ELISA plate reader at 405 nm (also if the reaction has been stopped with NaOH).

3. For detection of TMB, measure OD in a normal ELISA plate reader at 450 nm after the reaction has been stopped with acid (if the reaction is not stopped with acid, the colour is blue and can be measured at 655 nm).

Wash after detection to prepare for rehybridization or storage

1. Remove substrate from wells after measuring the catalyzed substrate.

2. Wash the empty NucleoLink wells three times, soak for 5 min, and wash three times with freshly made 0.2 M NaOH and 0.1% Tween 20 at RT.

3. Wash the empty NucleoLink wells three times, soak for 5 min, and wash three times with distilled water or 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20 at RT.

4. The washed NucleoLink Strips can be stored empty at 4° C or below in an ethylene bag. The Strips should not be sealed.

1. No rehydration of the NucleoLink Strips is necessary after storage. Commence with step 1 in the detection section, and add hybridization solution directly to the empty, dry, wells.