Can you attach the sequence .abi files for samples 1 and either 2or 3 please. It may give a clue as to what the problem is. One possibility is that the dna purification has left ntps, degraded dna,primer or rna in the nanodrop measured samples and nano measures all nucleic acids so will over estimate the amount of dna based only on absorbance values

Hello

In my experience, nanodrop may not give you accurate measures in lower concentrations (less than 10 ng/ul). The quality of your gel is excellent although there might be pipetteing inaccuracy.

I agree to Pauls' comment. Nanodrop measures all types of nucleotides in your sample (whole DNA, fragmeted sequences, oligos etc.) Therefore, the results are somewhat "unclear" related to uncertainties regarding purity of the sample. However, organic solvents or other compounts of utilized buffers etc. might also affect sequencing after isolation.

Hi Melissa,

For accurate results, a combined analysis of nanodrop, as well as agarose gel, is useful. I hope the gel image you have attached is the PCR product. The nanodrop quantifies nucleic acids and the dNTP left after PCR are also included. This can be a reason for non- uniform brightness of the bands. Also, make sure to mix the sample(by gentle tapping or by short spin) before loading to the nanodrop.