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Latest Questions
What could be the reason if annealed primer fragments cannot be ligated into a vector?
If the primers in the ligation reaction lack a 5' phosphate group, the ligation efficiency is relatively low, but it may not completely fail.
If the phosphorylated product still cannot be ligated into the vector, it is necessary to check the vector digestion efficiency and review the primer design. If no issues are found, consider optimizing the primer annealing conditions and the ligation reaction system.
Can the synthesized DNA/RNA product quantity be determined based on the brightness of the electrophoresis band?
No,Ethidium bromide (EtBr) stains nucleic acids by intercalating between the bases in double-stranded helices. Synthetic DNA/RNA molecules are single-stranded. They can only be stained by EtBr if they form local hairpin loop structures through self-folding or form partial double-helix structures between strands. Because different products have different sequences, their ability to form double helices varies, and consequently, their staining capacity is not uniform. Therefore, the brightness of the EtBr-stained band cannot be used to quantify synthetic DNA/RNA.