Agarose Gel Electrophoresis of DNA Amplicons Post PCR: A Method to Analyze Products of Multiplex PCR and Evaluate PCR Reaction Success

Protocollo

1. PCR Amplicon Confirmation by Gel Electrophoresis

1. According to the manufacturer's recommendations, prepare a volume of 1.5% (w/v) agarose gel in 1x tris-borate-EDTA (TBE) buffer appropriate for the number of PCR reactions to be tested. Prior to casting, add 5 µL of fluorescent nucleic acid dye per 50 µL of gel solution and mix. Use trays and combs as appropriate for casting, leaving an appropriate number of wells free for DNA reference ladders.
2. After setting, submerge the gel in 1x TBE-buffer in a GE system. Mix 5 µL of PCR product together with 2 µL of loading dye and transfer to gel wells. Add 5 µL of DNA ladder in empty wells for reference.
3. Run the gel at 110 V per 15 cm for approximately 1 h and use a UV-based gel imaging/visualisation system to verify the presence of multiple (up to five) bands representing PCR amplicons (see example in Figure 1). Discard the gel. Store remaining PCR products at 4 °C until further processing.

Risultati

Figure 1: Gel electrophoresis verifying the presence of multiple PCR products. The image confirms the presence of multiple PCR amplicons in all 12 lanes containing samples, with the first lane representing the DNA ladder used. The sizes of selected ladder fragments have been indicated, as have the PCR assay and strain affiliation of each lane.

Materiali

Name	Company	Catalog Number	Comments
Agarose, universal, peqGOLD	VWR	732-2789P/732-2788
Tris-borate-EDTA (TBE) buffer	NA	NA	Standard recipe; produced in-house.
DNA Gel Loading Dye (6X)	Thermo Fisher Scientific	R0611
Gel electrophoresis system	As preferred	NA
GelRed Nucleic Acid Stain, 10,000X in water	Biotium	41003	Fluorescent nucleic acid dye
GeneRuler 50 bp DNA Ladder, ready-to-use	Thermo Fisher Scientific	SM0373	DNA ladder
UV-based gel imaging/visualisation system	As preferred	NA