Technical Documents

	Tech Notes and Bulletins
	Cell Culture
	Molecular Biology
	Immunology
	Handling, Storage, and Transport
	Ordering Publications
	Cryopreservation Manual
	NucleoLink App. Guide
	FAQ's
	Technical Support 1-800-625-4327 nnitech@nalgenunc.com

PCR-ELISA Protocol

Revised version - April 1, 1998

Covalent binding of solid phase capture oligonucleotide

1. The solid phase capture oligonucleotide must be phosphorylated or aminated at the 5'-end and a linker of at least 10 T's (Thymidine's) should be added to the 5'-end of the oligonucleotide.

2. Prepare a freshly made coating mix consisting of: 100 nM solid phase capture oligonucleotide and 10 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) in 10 mM 1-methyl-imidazole (1-MeIm) (pH 7.0).

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

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 the empty NucleoLink wells three times, soak for 5 min and wash three times, all 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. The amplification should be made as normally in traditionally PCR tubes.

2. In order to label the PCR product, the amplification should be made using either
    

   1. Biotinylated primers (one or both PCR primers can be labelled) or

   2. Addition of DIG-11-dUTP (a digoxigenine labelled oligonucleotide) (Digoxigenine-11-2'-deoxy-uridine-5'-triphosphate, alkali-stable. Boehringer Mannheim, Cat. No. 1093 088) at a concentration of 4 µM. The concentration of dTTP should be lowered to 0.125 mM. All other concentrations are unchanged.

If non-labelled PCR products are detected, a labelled probe, complementary to the same strand as the solid phase capture probe, should be added during the hybridization. Other labels can also be used, but the above-mentioned labels have all been tested by the Nunc A/S research laboratory.

1. Add 10 µl of the PCR product to the NucleoLink wells with the solid phase capture probe covalently bound.

2. Add 10 µl of 1 M NaOH with 0,5 mg/ml thymol-blue. This liquid is dark blue.

3. Incubate for 10 min. at RT.

4. To each well, add 80 µl of 6.25 ´ SSC, 0.625% BR (Blocking Reagent, Boehringer Mannheim, Cat. No.: 1096176), 0.125 % Tween 20 and 0.5 M NaH₂PO₄ adjusted to pH = 6.5 with NaOH. The pH of this mixture is 7.5 and the liquid should become yellow. If the colour is red (acidic) or blue (alkaline), the hybridization will not be successful.

5. Incubate for 30 min - 2 hours at 50°C (each system should be optimized individually).

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

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

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

9. Detection of biotin-labelled PCR product:

1. When using Alkaline phosphatase (AP): Add to each well 100 µl AP conjugated streptavidin (Code no. D0396 - DAKO, Glostrup, 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.

10. Detection of digoxigenine labelled PCR product:

1. Add to each well 100 µl anti-dig conjugated AP (Anti-digoxigenine-AP, Fab fragments, Boehringer Mannheim, Cat. No. 1093 274) diluted 1:5000 in 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20, and 0.5% BR.

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

12. 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.

13. 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; TMB (3,3',5,5'-tetramethylbenzidine) in a ready-to-use solution (Cat. No.: 4380H. Kem-En-Tec, Copenhagen, 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.

14. Substrate incubation.

The NucleoLink Strips should be sealed with Nunc Sealing Tape when incubating for longer than 30 min.

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

2. When using pNPP, incubate at RT for 30 minutes (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.

15. To measure the signal.

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

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

   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).