10.3.3. Fragment analysis
The MLPA reaction products can be analysed on conventional slab electrophoresis, using a 4% agarose-TBE gel (De Smet et al., 2012; Green and Sambrook, 2012), or using a high-resolution, semi-automatic electrophoresis system such as the BioAnalyzer (Aligent), Experion (Biorad), Qiaxcel (Qiagen) or MultiNA (Shimadzu), which are designed for separating short fragments. In all cases, interpretation of the results is simplified by loading a specific MLPA ladder, generated amplifying each of the MLPA targets individually from cloned controls and pooling these into a single ladder.
- Agarose gel
1. Prepare a 4% high resolution agarose gel in 1x TRIS-Borate-EDTA buffer (Green and Sambrook, 2012).
2.1. 10 µl aliquot of the MLPA reaction,
2.2. 5 µl 4x Sample Buffer.
3. Load gel.
4. Run for 45-60 minutes at 75-90 volts.
high-resolution gel electrophoresis:
1. Use gel system appropriate for 25-500 bp DNA fragments.
2. Follow manufacturers’ instructions for sample preparation, loading, running and data analysis.