Hi guys,i've been having some problems with my standardized PCR protocol lately.I had a band in my negative control for some reason but amplification of samples was fine(i.e.positive samples showed bands and negative showed no amplification).I changed my vial of water and all was good.But the next time i changed to a different vial of water from the same batch, i again got the band.Any clues?
Also a major annoyance is that in this very PCR i loaded samples extracted using two methods.In one method i got results as mentioned earlier but in DNA samples extracted using phenol-chloroform method i got no amplification AT all -only sheared DNA.Is this because the DNA is degraded?or is it that the concentration was too high(around 500ng in 25ul mix?)-even though i have used higher concentrations earlier with success.
Id be grateful for any advice.
PS:My amplicon is 720bp