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PCR Amplification Preparation with Labeled Probes Protocol

Experiment Summary

The deoxy nucleotides in the PCR reaction system are partially replaced by labeled deoxy mononucleotides (usually labeled dUTP instead of dTTP), and the labeled groups are randomly adulterated into the amplification product-DNA probes after PCR amplification. This results in high probe concentration, high amount of detectable labeling groups, high sensitivity, convenience and simplicity compared to the notch shift method of probe preparation, and is particularly effective in detecting low abundance, low copy number target fragments due to high sensitivity. Generally, the labeled groups available for adulteration can be isotopically labeled groups, ozonated deoxyuridine labeled groups, or other non-released labeled groups such as biotin, digoxin, etc. The following is an example of PCR labeling of probes with biotin and digoxin.

Experimental Steps

1. Biotin labeling

Application of bio-11-dUTP as a partial replacement for dTTP adulteration in PCR amplification probes.

(1) Formulation of reaction system
Composition of dNTP dATP, dCTP, dGTP 20 mmol each, dTTP 15 mmol, bio-11-dUTP 5 mmol mixed, other reagents as in conventional PCR.

(2) After 25 cycles, freeze, take out the freeze, and absorb the paraffin oil while the lower water phase is still in the frozen state.

(3) 1 ul of 20 mg/ml glycogen in aqueous phase, pH 5.2, 2.5 M naAC 10 ul, 220 ul of anhydrous ethanol. Mix well and leave overnight at -20°C.

(4) The precipitate was taken by centrifugation, freeze-dried and then 100 ul of water or TE was re-dissolved.

2. Digoxin labeling

Application of dig-11-dUTP as a partial replacement for dTTP adulteration in PCR amplification probes.

(1) Formulation of reaction system
Composition of dNTP dATP, dCTP, dGTP 200 umol each, dTTP 130 umol, bio-11-dUTP 70 umol mixed, other reagents as in conventional PCR.

(2) 35 cycles amplification products
30 Amplification products were purified in the same way as for DIG randomly labeled probes (for precipitation only, 5 ul of 4MliCl and 150 ul of ice ethanol were added to 50 ul of PCR reaction solution).

Attention

1. The proportion of labeled groups in dNTP should not be too high when labeling, because the labeled groups have a site-blocking effect compared with dTTP, and the proportion is too high for PCR amplification and later hybridization to be affected.

2. The amount of target DNA should not be too high, controlled around 0.2 mol in biotin labeling; it can be as low as 0.1 ng in digoxin labeling.

* For research use only. Not intended for any clinical use.
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