So my first question is this:
I had isolated genomic dna.. ran the taq pcr.. isolated the one band.. purified it from the gel. Before I ran Big Dye PCR, nanodrop showed me 47.2 ng/uL. I ran a big dye pcr rxn with forward sequencing primers...
this PCR tube had: Ready Reaction Premix - 1Ál. Big Dye Sequencing Buffer - 3.5Ál. Primer - 1Ál. Template - 1Ál. Pure Water - 13.5Ál.
I ethanol precipitated this and then sequenced but got absolute crap (biochem professor didn't think it looked like noise but there were some very very low peaks). Any suggestions for what could have gone wrong?? I think maybe the pure water used had a contaminant or maybe I got rid of the pellet during ethanol precipitation?
My second question: I have genomic DNA and after I run taq PCR... I am not able to see any bands! The primers were designed with primer3 and our professor ok'ed them. I even ran a gel at 1.5% agarose to see if I could detect something (our usual is 0.8%).
My taq polymerase reaction reagents look like this:
Taq buffer (10x) - 5Ál
Deoxynucleotides Mix - 2Ál
Upstream primer - 2.5Ál
Downstream primer - 2.5Ál
DNA template - 2Ál
Taq polymerase - 0.5Ál
Nuclease Water - 35.3Ál
Both of the primers used for this have a Tm of like 61 so the standard protocol should be more then fine. Anyone have any suggestions? Currently the genomic DNA has a nanodrop reading of only 18 ng/uL which is kind of low. Could this most likely be my problem? Thanks!