spectrophotometry query - (Jun/21/2006 )

Hi,

I am busy analysing data from an experiment in which I compared 6 different extraction buffers.
Interestingly the amount of DNA measured with the spectrophotometer varied greatly between buffers eg. from 11 000 ng for one buffer to 400 ng for another, and all from a sample with the same number of organisms!!

I know that certain chemicals eg. phenol have an absorbance maximum close to A260nm and may artificially increase the readings. Are there any other chemicals, additives that do the same??

Thanks
Shaunb

-shaunb-

Loads. You will, of course, be doing a blank reading in which you have your buffer but no DNA sample?

-paraboxa-

Make sure that you are using the same buffer to blank with. We had problems for a while where the buffer that was being used to do the blank readings was a different lot number from the one that the DNA samples were in. Caused all sorts of variation in the readings.

-jamie419-

QUOTE (jamie419 @ Jun 22 2006, 07:14 PM)

Thanks I will investigate this further.

-shaunb-

QUOTE (paraboxa @ Jun 22 2006, 07:10 PM)

Loads. You will, of course, be doing a blank reading in which you have your buffer but no DNA sample?

Thanks for the info. Do you have any idea about certain chemicals such as B-mercaptoethanol, SDS, Triton X-100.

-shaunb-

Thanks for the info. Do you have any idea about certain chemicals such as B-mercaptoethanol, SDS, Triton X-100.
[/quote]

Fraid not right away. I think the Merck Index will have that info, though. Or there is a wonderful Biochemical research book that contains lambda max, pKa, pI etc info for just about everything. My copy is at home so I'll check it tonight for you. You could try running your own scanning absorbance on these reagents. I|t' snot just if these compounds absorbe but if they absorb at (or near) the wavelength you are using.
Maybe the manufacturers will be able to tell you over the phone.

Cheers.

-paraboxa-

ShaunB,

I checked up that Biochemical Research book. SDS and mercaptoethanol have no lambd amax..I'm not sure if that really means they wouldn't be measurable so much as they simply aren't listed. Dithiothreitol has a lambda max at 283nm. Depending on the bandwidth of your specy I reckon that would show up when you were doinbg 260/280 purity checks.

Hope that helps.

-paraboxa-

QUOTE (paraboxa @ Jun 27 2006, 11:01 AM)

Thanks for the info and follow up. I have decided to repeat the whole experiment. This time making sure I blank each sample on it's appropriate buffer and also perhaps testing different amounts of each of the buffer components to see what effect, if any, they have.

Regards

-shaunb-