My name is JC. I need help with PCR problem. Performed CDNA synthesis using Quanta Bioscience FLEXI.
Oligo dt primer (2ul) and Random primer (2ul) were used in the reaction. (the two primers were mixed together to create a master mix for 16 samples). Primer mix was left overnight.
Complete the CDNA synthesis next day and did PCR amplification with another specific set of primers for target site around 300bp.
50ul of pcr product was generated using 4ul of forward primer and reverse primer, + 25ul of 2xpromega mix+5ul of CDNA and made it up to 50ul with nuclase free water.
Ran electrophoresis with 1.5% agarose gel + 2ul of ethidium bromide.
No pcr product formed but huge primer dimer at around 100bp in the gel?
Could the primers in CDNA anneal when mix together.
Or has the RNA degraded- but RNA was extracted and stored -80 using qiagen RNAesy kit set. Extracted and stored at the same day and use 2 weeks after.
HELP-need some advice. I can't figure it out