(by silvergrin from gmail.com)
Wed Jul 18 12:20:17 EST 2007
Well in case anyone in the future has to do merlin maxiprep
troubleshooting, the problem seemed to be *with the old merlin III
buffer*. I checked the pH and it was a little high. I took some new
buffer III I'd made, adjusted the pH to 4.8 (the protocol I was given
for making the buffers said nothing about buffer III's pH >:P ), and
did the prep as usual and it worked beautifully!
Chatting; initially when our lab first started doing merlin maxipreps,
after the spin-down there would be a fine precipitate that made the
lysate look cloudy and was generally a pain to filter. It would clear
up when isopropanol was added, and I still got decent purity and yield
at the end. After a while the lystates began to be clear and
filtering was much easier...the assay still worked until this week.
When I repeated the prep with the new buffer III with pH 4.8, the
lysate was cloudy again! So, I guess that's actually a good sign...?
Or maybe my pH is too low; I've read some protocols calling for a pH
DK -- at the same time, I tried a prep using 1:1:1 of buffers I, II,
and III, along with 0.8v isopropanol. This worked too!
Incidentally, with this prep I used new merlin III without futzing
with the pH (it was 5.2), and the lysate was very clear and did not
have a fine precipitate.
(waves to search engines)
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