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Can you use same samples for real time pcr 2nd time?

Real time pcr

Best Answer Trof, 25 January 2015 - 05:49 AM

OK, I'm affraid you seriously lack a detailed knowledge of what is happenng in PCR. I mean really, if you know how PCR works in detail (if not, please google it) then try to think why this whole idea of reusing makes no sense at all.

 

You got probably the idea as I can see, but you need to be completely sure about such things next time, or it would be very difficult for you to work in molecular biology.

 

You were practically asking that if you cooked a diner at some temperature and was observing how the food changes from raw to cooked, if you can use it again, cook it at different temperature and observe the changes from raw to cooked again.. seem rediculous, right?

 

So that's the same with real-time PCR, once it runs, it's over. You can make a melting analysis on it, after, but that's it.

 

If you want to test different temperature of a PCR, you need to make everything again, same as you need to make everything anew when trying to cook diner differently. 

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#1 J092

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Posted 24 January 2015 - 12:04 AM

Hi guys, I made samples yesterday and ran through the real time pcr machine, I've got my original samples as in my CDMA, primers etc. I can make it all from scratch. However I'm a bit confused the real time pcr tubes I put into the machine - am I able to use those again to repeat my real time for second time? I would think not as sample has been degraded, annealed etc. so would I have to do everything from scratch? I would like to confirm so I can plan my day, thank you.

#2 Trof

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Posted 25 January 2015 - 04:37 AM

I'm sorry but I don't get it.

 

You run samples through the real-time PCR machine, that means you got the fluroescent curves and probably seen it to reach plateau. These are used to calculate Ct and that stuff you do real-time for.

 

Now are you really asking if you can run them again?

Like the sample that already amplified and you can't see amplification curve again?

 

Or you ask if you can reuse only the tubes (that contain so much DNA contaminating everything around the moment you open it in the PCR area)?

 

I don't really have a clue what are you doing and why. Can you explain?


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#3 Trof

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Posted 25 January 2015 - 05:49 AM   Best Answer

OK, I'm affraid you seriously lack a detailed knowledge of what is happenng in PCR. I mean really, if you know how PCR works in detail (if not, please google it) then try to think why this whole idea of reusing makes no sense at all.

 

You got probably the idea as I can see, but you need to be completely sure about such things next time, or it would be very difficult for you to work in molecular biology.

 

You were practically asking that if you cooked a diner at some temperature and was observing how the food changes from raw to cooked, if you can use it again, cook it at different temperature and observe the changes from raw to cooked again.. seem rediculous, right?

 

So that's the same with real-time PCR, once it runs, it's over. You can make a melting analysis on it, after, but that's it.

 

If you want to test different temperature of a PCR, you need to make everything again, same as you need to make everything anew when trying to cook diner differently. 


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon






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