[RASMB] Optics check

[John Correia]

Sounds like you are doing intensity scans of real samples?  1st try intensity scans in velocity mode on an empty cell at low speed (3-4K) or on an empty hole, air vs air, where you scan the whole cell from 5.8 to 7.2 cm.  Vary the wavelength for each scan.  Machine spec is 20% variation in linearity & is done at 280 nm.   Often the intensity drops off near the top ~ 5.85 cm which is a function of the alignment of the flash lamp - slight rotation can make it better although not by much with the new lamp design.  This is your baseline for intensity.  With a real sample vs buffer the sample will absorb more light & of course they will not overlap.  The log ratio is the absorbance of the sample & in your case is must be > 1.5 - 2.0 OD at that wavelength?

 
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 Dr. John J. "Jack" Correia
 Department of Biochemistry
 University of Mississippi Medical Center
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 Jackson, MS  39216
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 email address: jcorreia at biochem.umsmed.edu     
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>>> <mitrana at mail.utexas.edu> 04/05/07 11:12 AM >>>

Hi all
I did a intensity versus wavelength scan at three radial positions - 5.8, 6.5,
and 7.2 cm. At 6.5 cm, it was as I expected with a 230 nm peak (I ~ 10000
units) and half that at 527 nm. However, at 5.8 cm, the 230 peak was almost
zilch and only the 527 prominent. And at 7.2 cm, 230 peak had an intensity of ~
13000 units and 527 half that. However, the sample and reference did not
overlay. The sample intensity was virtually zero.

I am a little lost right now as to what is going on. Are there anymore tests I
can run to identify the exact problem?

Mitra


-- 
Mitra S. Rana
Graduate Student
Institute for Cellular and Molecular Biology
2500 Speedway, UT-Austin
Austin, TX-78712
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