prodes

to the tune of Bad Boys (when your transformations are not working):

Bad bugs, bad bugs
Whacha gonna do
whatcha gonna do
when I pour bleach on ya!!!

Use of PCR products as standards is not usually recommended for a longer storage. They can be used, but only really fresh, because they degrade on the ends in time. Other thing is that standards containing only the desired sequence amplify with different efficiency than gDNA, because in gDNA you have lot's of "dummy"  DNA present, that is not amplified. Diluting the standards in buffer containing 20ng/ul rRNA or other dummy fill-in can mimic this effect. I once used standards like that, but I cut the specific product from the gel, which should eliminate the presence of other than specified sequence.

A wild guess.. I was thinking some time ago about the Mg2+ concentration that can be relatively changed in the presence or absence of nucleic acid, which traps the Mg ions. In your DNA sample you have a DNA concentration, the diluted standard has many copies but little actual mass, so more free Mg2+. Could the different Mg2+ concentration change the Tm of your standards? I don't know, but I find this idea interesting.

Also you probably have too high efficiency due to the inhibition in the more concentrated standards, that skewes the standard curve.

Many things can affect DNA migration in gel. Check this page "Top 10 Fun Facts for DNA Electrophoresis" to see if there is anything that might have caused the abnormal migration. For example "On a polyacrylamide gel, DNA fragments having AT-rich regions migrate slower than other DNA fragments of the same size."

According to Fermentas troubleshooting, here is a list of things that may affect migration of DNA in gel:

Atypical migration due to different DNA sequence or structure. During high resolution electrophoresis DNA fragments of equal size can migrate differently due to differences
in DNA sequences. AT rich DNA may migrate slower than an equivalent size GC rich DNA fragment. DNA structures such as nicked, supercoiled or dimeric molecules will always show different mobility on gels compared to an equivalent DNA size standard.

Gel shift effect. The presence of DNA binding proteins in the sample, such as ligases, phosphatases or restriction enzymes may alter DNA migration in the gel or cause the DNA to remain in the gel wells. High salt concentration in the sample may also cause gel shift effects.


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