Help RNA purification and cetrifuge time

[fleetwood99]

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.

 

[Moonbear]

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?

 

[oswaler]

Hello,

Thank you for reaching out. I understand that you are experiencing a decrease in your 260/280 ratio and believe it may be due to DNA contamination, possibly caused by using a slower centrifuge with your RNA purification kit.

To address this issue, it is important to first confirm that the decrease in the ratio is indeed due to DNA contamination. If possible, you can perform a DNAse treatment on your samples and re-measure the 260/280 ratio to see if it improves. If it does, then you can be more confident that the slower centrifuge is causing the issue.

In terms of calculating the appropriate centrifuge time at 3,000xg to match the 50 minutes at 5,000xg, there are a few factors to consider. The most important factor is the type of rotor being used in the centrifuge. Different rotors have different capacities and maximum speeds, so the time needed to achieve a certain g-force will vary. Additionally, the type of sample tube being used can also affect the time needed for centrifugation.

Without knowing these details, it is difficult to provide an exact equation or calculation. However, a general rule of thumb is that a 10% increase in centrifuge speed (g-force) may require a 20% decrease in time to achieve the same level of pelleting. Based on this, you may need to run your samples for approximately 60 minutes at 3,000xg to achieve the same level of pelleting as 50 minutes at 5,000xg.

I would also recommend consulting with the manufacturer of your centrifuge and/or RNA purification kit for more specific guidance on adjusting your centrifuge time. They may have specific recommendations for their products.

I hope this information is helpful and wish you the best of luck with your experiments.

Best,