i have some problems I am very worried about
I had isolated some RNA and made cDNA, then when i measured the concentration of my cDNA it was insanely high (around 2000 ng/microlitre) but my boss said it was okay and i could go ahead with the PCR, he just told me to dilute it.
so i diluted it with water so that every microliter had about 100 ng. i went ahead and did my PCR.
now on my gel, nothing showed up. i ran it again, this time with a ladder and i also ran my cDNA to see if that would show up.
i ran a 0.8% gel and my ladder was completely streaked through, some bands were kind of visible but not nicely defined, and my cDNA row, there was a clump at the top of the gel, near the well, but it looked like it was streaking too. again, nothing showed up for my PCR samples, but at the very bottom of the gel, past where the dye stain was, there was a clump of stuff visible, for both PCR rows.
my question: is my cDNA streaking because the concentration was too high? this is what i think it was. i put in 2 microliters which was around 4000 ng, that's wrong right? can i use the diluted cDNA (i diluted in water)? if so how much? 100 ng?
and secondly, why is my ladder streaking like that? what can cause a ladder to streak?
and how do i fix it?
and thirdly, what is the clump at the bottom of my PCR lanes?!
i am in desperate need of help, please please please help!