Help RNA purification and cetrifuge time

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SUMMARY

The discussion focuses on RNA purification for RT-PCR cloning using the Promega Total RNA Kit, highlighting a decrease in the 260/280 ratio from 1.9-2.1 to 1.7, indicating potential DNA contamination. The primary variable affecting this outcome is the centrifuge speed; the original protocol requires 5,000xg for 50 minutes, while the new centrifuge operates at only 3,000xg. The user seeks to determine the equivalent centrifuge time at the lower speed to achieve similar results, noting past experiences where inadequate centrifuge speeds led to poor outcomes.

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  • Understanding of RNA purification techniques
  • Familiarity with RT-PCR cloning processes
  • Knowledge of centrifuge operation and g-force calculations
  • Experience with the Promega Total RNA Kit
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  • Research centrifuge speed conversion formulas for g-force
  • Investigate alternative RNA purification kits with lower centrifuge speed requirements
  • Learn about troubleshooting DNA contamination in RNA samples
  • Explore laboratory resources for access to high-speed centrifuges
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Researchers, molecular biologists, and laboratory technicians involved in RNA purification and RT-PCR who require insights into optimizing centrifuge conditions for effective RNA extraction.

fleetwood99
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I've been purifying RNA for RT-PCR cloning. I have been using the same type of kit (Promega Total RNA Kit). My 260/280 ratio has been between 1.9-2.1, which is good. However I just ran a new set of samples and the ratio is down to 1.7 which isn't so great. This might mean I have a lot of DNA contamination. The only difference in the protocol is the centrifuge, it is not as fast as my previous centrifuge. The protocol calls for 5,000xg for 50 minutes. The new centrifuge only runs at 3,000xg. How much time do I need to run these samples at 3,000xg to be the same as 5,000xg for 50 minutes? I thought since the radius was the same I would just run the samples 40% longer. I've looked everywhere for an equation please help.
 
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Increasing the time may not be sufficient. I've run into that problem in the past where I just didn't have a centrifuge available that reached the speeds required by the kits (not that particular kit, just illustrating the point here), and it just didn't work. I had to find a lab to work in that did have a centrifuge that reached the appropriate speeds.

And, while I assume you've already done so before asking here, just in case...have you tried this more than once to be sure it wasn't just a one-time fluke with bad yield?
 

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