Friday, August 18, 2006

Cell lysis (or lack thereof)

My poor RNA recovery has resurfaced, and it is exclusive to cells in MIV for 30minutes, with improved (but still lower) yields from cells in MIV for 90minutes. I went back through my old notes to find that previously when working with Hflu, I had speculated that MIV treated cells require more intense lysozyme treatment. For many months I have been working with E. coli and had continued on with the same protocol when switching back to Hflu, with the standard thought that "Hflu is a wimp - it falls apart if you look at it long enough". But maybe I'm wrong, maybe MIV treated Hflu is a tough nut to crack. I have been visually cheking my cell suspensions for clarity, which is usually a good indicator of lysis, so I really don't know what's up.

Today I will add more lysozyme and also split my pellets so as to load less cell material onto the RNA isolation columns. It's possible that a tough cell wall is not the problem, but that something else about the cell constituents of MIV treted Hflu is negatively impacting column performance. By diluting the cell prep and loading less material, I hope to dilute the inhibitor and ultimately improve RNA yield.

I had a good visit with the Sigma rep yesterday. We really like their DNA purification and isolation columns (half the price and twice the performance of Qiagen = 4x better), and he said they now have RNA isolation columns, so he's sending a sample for us to try. It turns out that Sigma makes the buffers that Qiagen sells in their kits, so it's not surprising that Sigma can make a column and undersell their competitors (who they also sell to)... Sigma really can't lose.


At 9:56 AM, Blogger LindsayAWilson said...

I love Sigma. Bring on the new kits.

At 5:06 PM, Blogger Heather Maughan said...

Hmm...maybe H. influenzae isn't such a wimp after all? I beg to differ. What can you expect to withstand when you have a genome that is less than 2 Mb? I think we should blame it all on Qiagen.


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