1) Immunocapture stage
- Coating buffer: 15 mM Na2 CO3 ; 35 mM NaHCO3 , and 3 mM NaN3 , per liter (pH 9.6).
- Extraction buffer: (20 mM Tris-HCL (pH 8.0), 138 mM NaCl, 1 mM PVP, 0.05% Tween-20, 3 mM KCl, and 3 mM NaN3 per liter (pH 7.4).
- PBS-Tween wash buffer: 138 mM NaCl, 1.5 mM KH2 PO4 , 8 mM Na2 HPO4 , 3 mM KCl, 0.05% Tween-20, and 3 mM NaN3, perliter pH 7.4)
- Antibodies
2) PCR stage
For a single reaction of 50 ul, the PCR components are:
- 20 mM Tris-HCl (pH 8.4) (included in 10XPCR buffer depending on manufacturer)
- 50 mM KCl (included in 10XPCR buffer depending on manufacturer)
- 1.5 mM MgCl2
- 0.2 mM dNTP
- 50 pmoles of each primer (degenerate primers can be used)
- 1% Tween-20
- 2.5 U Taq DNA Polymerase
Method
(A) Preparation of clarified extracts:
Wash fresh foliar tissue briefly in sterile distilled water. Weight out 1 g and cut into strips with sterile scalpel blade. Grind tissue using autoclaved mortar and pestle (or extraction pouches) in extraction buffer (1X working conc.) at a ratio of 1:3 w/v at room temperature. Filter extracts through mira cloth (not required if using extraction pouches). Serially dilute extract to 20 to 2-10 in extraction buffer � use 2-5 and 2-6 dilutions for the antigen capture steps.
(B) Antibody coating steps
Dilute antibody 1:500 in coating buffer (1Xworking conc.)and mix gently by inversion. Aliquot 50 ul into 0.5 ml microcentrifuge tube. Place tube in a moist chamber. Incubate (see section (D) Varying duration times of protocol)
(C) Antigen capture steps
Pipette out diluted antibody Allow tube to air-dry (10-15 min) Aliquot 50 ul PBS-Tween wash buffer (1X working conc.) Pipette out wash buffer Repeat twice Allow tube to air-dry (10-15 min) Aliquot 50 ul of diluted plant extract Place tube in a moist chamber Incubate (see section (D) Varying duration times of protocol)
(D) PCR amplification
Pipette out diluted antibody Aliquot 50 ul PBS-Tween wash buffer (1X working conc.) Pipette out wash buffer Repeat twice Allow tube to air-dry (10-15 min) Aliquot 50 ul of PCR reaction mix
Perform your own PCR or conduct as recommended here:
For a single reaction of 50 ul, the PCR components include 20 mM Tris-HCl (pH 8.4), 50 mM KCl; 1.5 mM MgCl2 , 0.2 mM dNTP, 1% Tween-20, 2.5 U Taq DNA Polymerase and 50 pmoles of each primer (degenerate). Overlay reaction mix with PCR-grade, sterile mineral oil and subject to amplification using a programme of: 5 min at 94o C, followed by 40 cycles of 1 min at 940 C, 1 min at 550 C and 2 min at 720 C with a final extension of 5 min at 720 C.
(E) Varying the protocol duration time
IC-PCR Short Protocol (1 day single tube assay)
1) Antibody coating steps:
- Dilute antibodies 1:500 in coating buffer (1Xworking conc.)
- Incubate at 370 C for 2.5 h in moist chamber
- Wash 3X with PBS-Tween wash buffer (1Xworking conc.) �let air dry
2) Antigen capture steps:
- Grind leaf extracts 1:3 w/v in extraction buffer (1Xworking conc.)
- Incubate at 370 C for 2.5 h in moist chamber
- Wash 3X with PBS-Tween wash buffer (1Xworking conc.) �let air dry
3) Run PCR
IC-PCR Long Protocol (3 day single tube assay)
1) Antibody coating steps:
- Dilute antibodies 1:500 in coating buffer (1Xworking conc.)
- Incubate at 40 C for 16 h in moist chamber
- Wash 3X with PBS-Tween wash buffer (1Xworking conc.) �let air dry
2) Antigen capture steps:
- Grind leaf extracts 1:3 w/v in extraction buffer (1Xworking conc.)
- Incubate at 40 C for 16 h in moist chamber
- Wash 3X with PBS-Tween wash buffer (1Xworking conc.) �let air dry
3) Run PCR
