Does PEG affect A260 readings on a nanodrop? - (Mar/04/2008 )

Hello,

I am wondering if anyone ever encountered my problem.

I have tried to use PEG for some of my plasmid preps. I was surprised because when I compared my regular plasmid prep with alkaline lysis and RNase treatment to a plasmid prep (on the same original sample) using alkaline lysis and PEG, I am getting much lower A260 readings (1:10th of what the regular plasmid prep give me or even lower). I use the nanodrop to determine my concentrations.

I always run a 3 uL aliquot of my plasmid preps on a gel to check the plasmids and was even more surprised to see that the samples extracted with my regular protocol and the PEG protocol looked exactly the same: on the gel, it didn't seem like there was any difference in their concentrations.

Does anybody know if PEG affects A260 readings with the nanodrop? Are there other chemicals that affect A260 readings?

Thanks!

Lolo146

did you blank with the peg buffer?

maybe some peg coated the optics and reduced the response? try cleaning the pedistals.

After the PEG precipitation, I get a pellet that I wash with 70% ethanol, then I dry the pellets and resuspend them in TE. I am assuming I don't have any PEG left in my samples when I do the nanodrop.
We tried to add very small amount of PEG to some DNA samples with known concentration and it didn't seem to affect the A260 readings.
We cleaned the pedistals of the nanodrop several times and we still get a huge difference between the readings of the nanodrop and what we see on the gel.
Thanks!

have you tried reading the samples in a different spectrophotometer (not necessarily a nanodrop)?

how old (and used) is the nanodrop? you may be seeing signs of failure of the light source, optical fibers, calibration and/or photomultiplier. you may require service.

I didn't try reading the samples on a different spectrophotometer but I did test our nanodrop with samples of known concentration and it seems to be working just fine. We bought that nanodrop about a year ago so it it not very old.