PCR technology topic: immuno-PCR series three - basic experimental steps

Main program

(1) antigen + biotinylated antibody → antigen-biotinylated antibody complex;

(2) avidin→antigen-biotinylated antibody-avidin complex;

(3) adding biotinylated DNA→antigen-biotinylated antibody-avidin-biotinylated DNA;

(4) PCR amplification of the biotinylated DNA fraction.

Experimental procedure

(1) Preparation of biotinylated DNA

The phagemid BLuescript skt was amplified by PCR with biotinylated M13 primer to prepare a 280 bp DNa fragment containing T3 and T7 primer sequences, which is biotinylated DNA.

(2) ImmunoPCR mode

Reagent

Coating buffer: 20mmol / L Tris-HCI (pH 9.5), containing 150mmol / L NaCl and 0.02% NaN3;

Washing liquid: 20mmol / L Tris-HCI (pH 7.5), containing 150mmol / L NaCl 0.1mmol / L EDTA and 0.1% Tween20;

Diluent: 20 mmol/L Tris-HCI (pH 7.5) containing 150 mmol/L NaCl 0.45% skim milk and denatured salmon sperm DNA (0.1 mg/ml).

2. Operation

(1) Coating

The antigen (BSA) was diluted with a coating buffer, added to a microtiter plate (45 μl well), and left overnight (about 15 h) at 4 ° C, and washed with a washing solution 3 times × 5 min.

(2) Closed:

Add 200 μl of 25% skim milk and denatured salmon sperm DNA (1 mg/ml), 0.1 mmol/L EDTA in 20 mmol/L Tris-Hcl (pH 7.5) per well (containing 150 mmol/L NaCl buffer, incubate at 37 ° C). 80min, wash the plate several times.

(3) Antigen-antibody reaction:

The monoclonal anti-BSA antibody was diluted 1:8000 with the release solution, 50 μl per well, 45 min at room temperature (22 ° C), and the plate wells were washed 15 times × 10 min to remove unbound antibody molecules.

(4) Chain affinity protein A binding reaction:

50 μl of the streptavidin-protein A chimera bound to biotin-pUC19 diluted with the dilution solution was added to each well, and the chimeric-pUC19 was bound to the solid phase antigen-antibody complex at room temperature (22 ° C) for 50 min. The plate was then washed 15 times x 10 min and washed 3 times with TBS without NaN3, and then the microtiter plate was used for the subsequent PCR reaction.

(5) PCR

Experimental conditions 50 mmol/L KCI, 10 mmol/L Tris-HCI (20 ° C pH 8.3), 1.5 mmol/LMgCl 2 , gelatin (10 μg/ml), 0.8 mmol/L dNTPs (0.2 mM each), 2 μM primers (each primer) 1 μM) and Taq DNA polymerase (50 U/ml).

PCR cycle:

Before the PCR, the reaction mixture was irradiated with ultraviolet (UV) (254 nm), and then added to the wells of the microtiter plate, 40 μl per well, and 20 μl of UV-irradiated paraffin was applied, and the PCR cycle was performed on the PCR machine at the following temperature. : Initial denaturation 94 ° C 5 min; 30 cycles: denaturation 94 ° C 5 min, annealing 58 ° C 1 min, extension 72 ° C 1 min, and finally extended 72 ° C 5 min, the resulting PCR product was a specific 261 bp fragment.

Reference flow

1. Dilute the bacterial solution to a certain concentration with 0.85% NaCl;

2. Add 50 μl to the microplate and overnight at 4 °C. At the same time set a negative control (add 50μl 0.85% NaCl in another well);

3. Wash with 500 μl of 0.05% warm 20pBS (hereinafter referred to as TPBS) for five times;

4. Add 2.25% of 100 μl normal goat serum (NGS) in PBS for 2 hours;

5. Wash 3 times with TPBS containing 0.15% NGS;

6. Add 100 μl of monoclonal antibody (containing 100 μg of fresh fish sperm DNA) diluted appropriately with 0.75% NGS and incubate for 30 min at room temperature;

7. Wash five times with TPBS;

8. Add 50 μl of the appropriate amount of biotinylated anti-mouse IgG antibody and incubate for 30 min at room temperature;

9. Wash five times with TPBS;

10. Add 60 μl of diluted biotinylated DNA and avidin and incubate for 30 min at room temperature;

11. Wash five times with TPBS and wash three times with HPLC grade water;

12. PCR amplification: 50 μl of PCR reaction solution (containing T3 and T7 primers), 95 ° C for 60 sec, 50 ° C for 110 sec, 72 ° C for 110 sec, 30 cycles. 10 μl of the PCR product was electrophoresed on a 1.7% agarose gel, and compared with the molecular weight standard of the nucleic acid, the electrophoresis band was positive at the corresponding position. The electrophoresis results are photographed as necessary for quantitative analysis.