User:Brigette D. Black/Notebook/Brigettes Notebook/2009/08/25/New Nanodrop Fail & Pi standard solution

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The new (and officially our) NanoDrop 2000c came in yesterday. I tried setting it up, but kept receiving an error upon opening the program. It would display "Optical switch initialization has started" then display an error message, "Error 1042: Low signal encountered during initialization. Ensure measurement pedestals are clean and the instrument arm is down". It will then try to initialize the optical switch again, then display the error again, on and on.

I ensured the the pedestal was clean 5 times by cleaning it with 5 uL DI water (just as described in the manual), blew some air into the cuvette holder (just like the manual says), and the message continued. After a lot of exiting, restarting, switching users, etc, Lihn installed the software on his computer, and it still didn't work. So I am pretty sure this is the fault of the hardware and I think it is really broken. I emailed the Thermo Scientific tech support and customer service centers, hopefully they will get back to me soon with either the magic thing to do that will fix it instantly, or they can send us a new one.

So with the broken NanoDrop, I went back to an old problem I was having before I took off for prelims. We are almost out of the "1mM Phosphate Standard" solution that comes with the PhosFree assay from Cytoskeleton. Various sources lead me to believe that this was infact simply 1mM monopotassium phosphate. I had tried to make some more in July, but my measurements were completely screwy and about 10 times higher than they should have been. Today I went back and looked at my notes from when I had made the stock solution, and realized that I had actually mislabeled the stock solution of monopotassium phosphate I had been using (50 uM instead of 500 uM). The label is now changed, it is actually 500 uM stock, I have spectrophotometry measurements to prove it, so all is good and well now in label-land.

So to make the phosphate standare replacement, I made 10 mls of a 1 mM monopotassium phosphate solution from the 500 uM stock (20 uL monopotassium phosphate @500 mM + 9.98 mL DI H20). I then ran it through a 0.2 uL filter.

Now, I am super prepared for the NanoDrop to be fixed! (ps, it is still looping between initializing the optical switch and displaying the error 1042).

Ramalldf 03:45, 26 August 2009 (EDT):Hi Brigette, I saw that you guys just got a nanodrop (s?) machine and since we were considering getting one, I was curious to know if you had done any experiments to see how closely the readings matched those of a regular UV-Vis spectrophotometer. I used the one in the Osley lab's building last year, but never had a chance to run any experiments myself...... (Oh nice to meet you by the way, I'm a Koch lab alumnus I guess).

Brigette D. Black 05:00, 26 August 2009 (EDT): Hello! It's nice, as well as somewhat surprising, to see someone is reading this stuff! I did not do very many measurements using the UV-Vis, although the one good measurement I got from that machine I did repeat with the NanoDrop. I did notice that the NanoDrop reports absorbances about 10% higher than the UV-Vis (the discrepancy at lower absorbances seems to be less). The machine seems very self consistent though, so the discrepancies aren't a symptom of error. I should mention that I have only done cuvette measurements, so I can't give any comparison for the pedestal. If you want to see the actual distinctions, I have the standard curves on this notebook page. Hope this helped you! Good to meet you!