Terrible PCR run

[studentresearcher]

Hi,

First time posting on this website. I am a student researcher studying the effects of meth on various mRNA targets. I work in a psych lab that primarily has done behavioral neuroscience research and is trying to get into more molecular level diagnostics. After finding and establishing protocols for the lab, I have finally done my first PCR run, using a Bio-Rad iCycler iQ RT PCR Detection system....Basically it turned out terrible and I could use some help determining how to troubleshoot from here..

All input is much appreciated.

[butters]

Are you using in-house self prepare PCR master mix? I think you may have over added some component.

but if you are not. I need more information.

[studentresearcher]

I appreciate your response. I am using a master mix from applied biosystems along with my mRNA fluorescin probe from the same company. This run utilized tissue punches from rat brain tissue that was extracted via Phenol–chloroform extraction technique. I converted to cDNA via iScript cDNA synthesis kit. The mRNA target was the housing keeping gene GAPDH.

Any other info needed?

Thanks again!

[nicol]

Did you check for RNA quality?

[davidflora]

Decrease annealing time
Increase annealing temperature
Decrease extension time
Decrease extension temperature to 62-68º C
Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM.
[Only registered users see links. ]