So I'm new to research but I am very thorough and I feel as though I have good technique. I've been doing lots and lots of PCR recently, and I usually get good results. However, this past week I have been having some major issues. I am setting up my reactions (25 uL total) as follows:
Component Per tube (uL)
10X PCR buffer 2.5
50 mM MgCl2 1.0
10 mM dNTP 0.5
Fwd primer 1.0
Rev primer 1.0
Taq (5,000 U/mL) 0.25
Sterile H2O to 24.5 uL + 0.5 uL template
I usually use 1-1.5% agarose depending on the size of my products, and lately I've been running at 80V in 1X TAE. My problem is, when I view my gels my "bands" are just huge vertical streaks. I don't think any of my reagents are contaminated, as I'm very careful. I've also used the primers before so I don't think that's the issue either.
I was thinking my template was degraded, but what's even more strange is that the bands in my ladder, which is brand new, come out crooked and with terrible resolution. If I use a ladder in a lane closer towards the middle of my gel, the entire ladder comes out curved. I've been making fresh gels every day and I give them plenty of time to set. What I have noticed is that my gels have been coming out of the gel box unusually warm.
Please help me figure out what's going on here. Is my template degraded? Buffer no good? Power supply no good? All of the above?
Edited by Chops369, 04 August 2014 - 04:29 PM.