How to validate qRT-PCR primers
Discussion:
https://www.researchgate.net/post/How_to_align_designed_PCR_FW_REV_Primers_with_DNA_sequence_Which_software_is_best_to_use
Information to garner per primer set:
1) # Possible amplicons with < 3 mismatches (via NCBI Primer blast)
2) Amplicon size (via NCBI Primer blast)
3) Whether pair spans intron (va UCSC in-silico PCR or BLAT)
UCSC
BLAT - determine total number of possible amplicons, amplicon ID, visualize track alignment to determine if spans intron
in silico PCR -
1) optimal PCR conditions
2) Amplicon length
3)
1) Check total possible amplicons produced
Enter Primer_F and Primer_R sequences into NCBI primer blast
https://www.researchgate.net/post/How_to_align_designed_PCR_FW_REV_Primers_with_DNA_sequence_Which_software_is_best_to_use
Information to garner per primer set:
1) # Possible amplicons with < 3 mismatches (via NCBI Primer blast)
2) Amplicon size (via NCBI Primer blast)
3) Whether pair spans intron (va UCSC in-silico PCR or BLAT)
- If one primer does not align, but NCBI reports mRNA product, usually means 1 primer spans an intron.
UCSC
BLAT - determine total number of possible amplicons, amplicon ID, visualize track alignment to determine if spans intron
in silico PCR -
1) optimal PCR conditions
2) Amplicon length
3)
1) Check total possible amplicons produced
Enter Primer_F and Primer_R sequences into NCBI primer blast