|Reverse Transcription PCR
||[Jul. 26th, 2011|11:51 pm]
Why does my RT-PCR suck? Because the RT-extension primer has a nasty hairpin Tm one degree below the protocol's RNA-to-DNA temp. Why didn't I know this? I assumed the previous worker checked the primer design before placing the order. Why didn't using the alternate primers help? Because they were designed by a second person who hadn't checked their design for secondary structures. What's the lesson? Don't assume anything's been checked unless the analysis is written down. |
The good news is, now I know why my experiments were broken, and I'm getting up to speed on primer design damn fast.