To what extent does the 260/230 value affect the efficiency of PCR reactions ? Do the constituents leading to a low 260/230 value (EDTA, guanidine salts and oligosaccharides) inhibit PCR ? I have been getting pretty low values of 260/230 (0.5-1.0) for DNA extracted using Charge Switch Protocol using Magnetic beads and the PCR has been failing. Also, is there any reason why I might be getting low 260/230 values in Charge Switch method ? (I was getting good values using Phenol-Chloroform extraction method)
EDTA carbohydrates, phenol all have absorbance near 230 nm. TRIzol reagent is a phenolic solution which absorbs in the UV both at 230 nm and ~270 nm
Guanidine HCL used for most DNA isolations will absorb at ~230 nm also
All these things will seriously inhibit your pcr, (and lower your ratio) especially the denaturing agents like guanadine.
All these DNA kits are different spins of a pretty similar process.
If you have a good steady centrifuge stick to the phenol & ethanol. Be certain you are drying your pellet well, but don't over dry it. I'll hear my pellet at 37 for 3-4 min to get rid of ethanol. Ethanol no good in pcr. If you hate doing ethanol precipitation (which I do) cause of invisible pellets, add a high grade bit of glycogen to your aqueous phase before the precipitation. This will make your pellet visible and is completely inert.
If your cleanup isn't the problem do an annealing and Mg gradient and up your primer quantity.
May want to give me more info like the individual absorbance, pcr conditions, template size etc
Traditionally bead purifications gives really good purity, reason I use it for microarrays. But it's expensive and not necessary for pcr.
Charge switch is just changing the charge of the bead by changing the ph.