Hello everyone，
I am developing asymmetric RT-RPA amplification to obtain the ssDNA products. The outputs of the ssDNA still are very low although I tried to various ratios of excessive and limited primers. How can I increase the ssDNA yields?
Kais Khudhair al Hadrawi
Optimize Reaction Conditions: Adjust temperature, pH, and magnesium ion concentration for enhanced efficiency. Optimize Primer Design: Design primers for efficient binding, considering length, GC content, and specificity. Increase Template Concentration: Boost RNA or cDNA concentration, but be cautious of nonspecific amplification. Optimize Reverse Transcription: Ensure efficient RNA to cDNA conversion with quality enzymes and conditions. Use Hot-Start Polymerases: Enhance specificity by inhibiting polymerase activity until optimal conditions are met. Include Stabilizers and Enhancers: Additives like DMSO or betaine can improve reaction efficiency and specificity. Reduce Inhibitors: Minimize contaminants and inhibitors for improved amplification. Optimize Reaction Time: Find the ideal incubation period for maximum ssDNA yield. Consider Pre-amplification Steps: Use techniques like rolling circle amplification to increase ssDNA before RT-RPA. Maintain Quality Control: Regularly check the quality of components to ensure successful amplification.
To summarize strategies for increasing single-stranded DNA (ssDNA) yields in asymmetric reverse transcription-recombinase polymerase amplification (RT-RPA):
Implementing and refining these strategies can optimize ssDNA yield in your RT-RPA reactions.
1 votes
0 thanks
- Badges
- Science topic
More Lin Huang's questions See All
Similar questions and discussions