I am doing reverse transcription using a semi-suppressive PCR therefore I introduce a template switching oligo at the 5' end of my transcript which is complementary to the PCR handle at the 3' end. Problem is, that I cannot separate some of the transcripts using specific 5' primers and the 3' handle primers for them because I get non-specific amplification. I cannot change the 3' handle so I was thinking to mutate the template switching oligo and design a primer accordingly. Does anyone have an idea about how this could be done as conservative as possible? For example, which end or nucleotides I should target?
Dear Theodore Kapellos
Please you can read the article titled ((Mutate a DNA sequence)) in the following link:
https://teach.genetics.utah.edu/content/dna/MutationInstructuctions_16-10-18.pdf
Hope to be benefit. Regard
Before introducing mutations, have you considered doing minor changes to one or both of your primers, for example making them 1 or 2 nts shorter, or perhaps longer with higher annealing temp to see if you can reduce non-specific amplification.
- Badges
- Science method