BSP Clone Sequencing

Summary

Methylation testing of DNA

Principle

Genomic DNA was treated with bisulfite, and all unmethylated cytosines were converted to uracil, while methylated cytosines remained unchanged; then primers were designed at both ends of the CpG island for PCR, and the target product was purified and then subjected to TA cloning, and the positive clones were picked for sequencing in each clone, and finally, the sequences sequenced were compared with the original sequences, and the methylation sites and numbers were counted and the degree of methylation was analyzed.

Appliance

Methylation Detection

Operation method

BSP clone sequencing method

Materials and Instruments

[Material] DNA target fragment;
[Reagent] Bisulfite;

Move

Genomic DNA PreparationBisulfite ModificationDNA purification and recoveryPCR AmplificationProduct CloningSequencing

Caveat

1, Tissue samples: -20℃ or -80℃ kept in a low-temperature refrigerator, transported by liquid nitrogen or dry ice; ice packs are available for intra-city or short-distance transportation. If the materials provided are fresh tissues, blood cells and other biological materials, please provide a sufficient amount of material to extract more than 2ug of gene DNA. 2, DNA samples: volume ≥ 20ul, concentration ≥ 100ng/ul, total amount of DNA ≥ 2ug, stored at 20℃, low temperature ice bag transportation. 3、Blood samples: required to be preserved in anticoagulation tubes or frozen storage tubes, volume ≥2ml, please use dry ice for transportation to ensure that the DNA is not degraded. 4、Samples for paraffin-embedded tissue sections, require the provision of 10 tissue sections of light film (area > 10mm × 10mm, thickness of about 5-10um)

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