PCR products electrophoretic silver staining assay

Summary

There are two commonly used nucleic acid electrophoresis methods. ① Horizontal electrophoresis: agarose gel, bands are judged under UV light. This method is economical and time-saving, but the resolution is low. ② Vertical electrophoresis: polyacrylamide gel, and the bands are judged under visible light. This method is relatively laborious, but the resolution is high. The high resolution is the key to the prioritization of polyacrylamide electrophoresis for the determination of clonal gene rearrangement bands. Bands that are judged to be false-positive (negative) on polyacrylamide gels that are wider and diffuse are often judged to be positive on agarose gels due to their poor resolution. Contents of "Immunohistochemistry Laboratory Techniques and Applications

Operation method

polyacrylamide gel method

Principle

Polyacrylamide gels (PAG) are polymerized from acrylamide monomers and the cross-linking agent methylenebisacrylamide, in the presence of free radicals provided by the initiator amine persulfate (AP) and the catalyst tetramethylethylenediamine (TEMED). The polymerization of acrylamide can be induced by natural light, so its aqueous solution should be stored in a brown bottle at a low temperature of 4℃. Because of the poor stability of the aqueous solution, it is usually better to use it up within 2 months. 10% amine persulfate (100 mg in 1 ml ddH2O) should be stored at 4°C and used up within two weeks. Polyacrylamide gel concentrations and DNA isolation and indicator display ranges are shown in Table 13-3. 12% polyacrylamide is preferred because the PCR products of IgH and TCR are in the range of 50 to 250 bp.

Materials and Instruments

PCR products
Ethanol Silica gel Polyacrylamide gel ddH2O Sampling buffer Tris Alkali Boric acid Nitric acid
Gel-filling glass plates Gel-making racks Fixed racks

Move

Preparation of 30% polyacrylamide: 29 g of acrylamide, 1 g of methylenebisacrylamide (the molecular ratio of acrylamide to methylenebisacrylamide is 29:1), add ddH20 to 100 ml, protect from light, and store at 4℃ (acrylamide has neurotoxicity, so avoid respiration into the body).

1. Glue making

1.1 Filling glass plate cleaning detergent washing, tap water rinse, distilled water rinsing, drying, ethanol soaking, wipe dry standby or coated with silica gel.

1.2 Spacer long glass and short glass plate pair into the glue-making rack, tighten the glue-making rack, and then put the glue-making rack on the fixed frame clamp clamping, pour the now prepared 12% polyacrylamide gel solution (ddH2O 48 ml, 10 × TBE 10 ml, 30% acrylamide 40 ml, 10% ammonium persulfate 1.85 ml, tetramethylethylenediamine 0.15 ml), insert the comb, do not make the Insert the comb, don't let the air bubbles under the teeth of the comb, and it will solidify in about 10 minutes.

1.3 After solidification, lift up the comb vertically and carefully, release the fixing frame, take down the gel-making frame, loosen the gel frame, and then take down the gel glass plate; use a syringe to suck distilled water to rinse the hole compartment, in order to prevent the unpolymerized solution from dissolving into the hole compartment and then polymerizing again after the comb is taken out and make the bottom of the holes not neatly shaped, which will lead to the electrophoretic band not being neatly shaped. Then fix the gel glass plate into double and face to face on the gel transfer rack and put it into the electrophoresis tank. The electrophoresis solution in the tank is 1 × TBE.

[10 × TBE electrophoresis buffer preparation: 108 g Tris base, 55 g boric acid, 40 ml 0.5 mol/L EDTA (pH 8.0) with water to 1 L; stored at room temperature].

2. Electrophoresis

2.1 Voltage 1～8 V/cm, too high a voltage will cause warming, leading to DNA band bending or even DNA unraveling of small fragments. The current should be 90-180 mA.

2.2 Sampling 4 μl of PCR product + 1 μl of sample buffer (0.25% bromophenol blue, 0.25% xylene cyanine FF, 30% glycerol in water; stored at 4°C) was mixed and pipetted into the wells with a fine pipette tip, and the relative molecular mass of the DNA was determined by using pBR 322 DNA/HaeIII (8-587 bp) as the Marker. 1 μl of Marker + 1 μl of sample buffer. 1 μl Marker + 1 μl Marker + 1 μl Marker + 2 μl 1×TBE were mixed and punched into the wells. The termination time of electrophoresis was determined according to the migration distance of the indicator contained in the sampling buffer. After stopping the electrophoresis, take out the glass plate and pry open the long and short glass plates gently with the spatula, separate the gel gently with the spatula, put it into the glassware containing _ddH2O, wash it for 2 times, and pour off the _ddH2O.

3. Silver staining

The following steps were performed on a shaker.

3.1 Fixation: inject 10% ethanol into the glassware containing gel after washing for 2 times, fix for 10 minutes; pour off the fixing solution; wash with _ddH2O for 2 times, pour off the _ddH2O.

3.2 Decolorization: Decolorize with 1% nitric acid for 10 min, pour off the decolorizing solution, wash with _ddH2O for 2 times and pour off the _ddH2O.

3.3 Staining 0.2% AgN03 solution for 20 min, pour off the staining solution, wash with _ddH2O for more than 2 times and pour off the _ddH2O.

3.4 Color development Add color development solution until the band is considered satisfactory, wash with _ddH2O for 2 times, rinse with tap water or store in 10% ethanol acetate termination solution. (Color development solution: _Na2CO3 30 g, formaldehyde 1 ml, 1% _Na2S2O3 0.1 ml, add _ddH2O to 1 L; store at room temperature.)

3.5 Archiving Gel recording system or digital camera to take pictures for archiving.

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