rtPCR working but the elongated product wont gel! - (Jun/01/2012 )
Hopefully someone can shed some light on my problem. I have been screening for a discreet virus using a 1-step mastermix and taqman probe. The taqman results are ok with ct values around 28 in positives and nothing in negatives. The problem i have is i wanted to check it was working exactly how i was expecting by running the product on a gel, which would also allow me to do absence/presence without using the probe.
To do this, i added a 15min elongation step at 68C and placed the product (after confirming the samples provided a Ct value) in a 1% agarose gel with EtBr. When i came to visualise the bands the only thing present was my ladder! I tried to repeat and noticed as i was pipetting into the wells it looked like the clear product was moving straight out of the wells and whilst i didn’t understand this, i assumed that would be the problem. The next time i mixed a little green buffer (used for standard PCRs) in with the rtPCR product and that went into the wells ok. The resulting image though again didn’t show any bands.
Does anyone have any ideas?
I might have this wrong, but are you saying you just loaded the product direct to the gel the first time, with no loading buffer? Coz that would be your problem. And second time around, you mixed it with some SYBR? I don't know what mix you have or if that has any glycerol or anything in it to help sink the DNA into the wells.
Try proper loading buffer.
What is your ladder made up in?