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RT-PCR carry over contamination and dUTP/UDG

RT-PCR UDG Uracil

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4 replies to this topic

#1 Rnotk

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Posted 20 March 2014 - 09:00 PM

I am bit confused about the use of dUTP and UDG (uracil glycosylase) for RT-PCR,

 

I know that there is a protocol to contain dUTP with dNTP for PCR and treat with UDG to prevent the carry over contamination, and I do understand the mechanism about how UDG works.

 

But my question is I still do not understand the importance of this methods. where the carry over comes and how it affects the result. 

 

Does anyone give me good practical example for this??? or Am I missing something?????

 

Thanks in advance



#2 phage434

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Posted 21 March 2014 - 07:16 PM

Very small amounts of amplified product can completely overwhelm the sensitive detection of DNA in a PCR reaction. People go to great lengths to isolate the amplfied reaction from the input DNA. This technique makes the amplified product unsuitable as a template for a subsequent PCR reaction, removing some of the concern about isolating product from template.



#3 Rnotk

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Posted 22 March 2014 - 12:27 PM

Thanks for the reply phage!

 

Yes, I do understand that concept, but what I dont understand is that we amplify the gene of interest for downstream experiments.

 

so in what instance, we need to remove amplicon and analyze original template??

 

personally, I never amplify template for RT-PCR. I just make cDNA and run realtime, so I do not know what other people do with this enzyme.

 

so help me to understand this!!



#4 phage434

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Posted 22 March 2014 - 05:52 PM

The problem arises when you (say) ampllify the GAPH gene from your sample, and determine the copy number of that gene on Monday, then you accidentally contaminate your pipettor with a little of that sample.  Then, Tuesday, you repeat your experiment, perhaps with a different sample. The very small amount of contamination on your pipettor looms very large when mixed into your Tuesday sample, and the copy number will be wildly off.



#5 Rnotk

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Posted 29 March 2014 - 08:29 PM

Thank you for the reply again.

That's actually what I thought initially, but I just wanted to make sure since I though there might be some special experiments which require the digestion of the amplicon for whatever the reason.

 

Thank you again and sorry about the delay of my response.







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