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primers with fluoresent dye PCR cycle question


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4 replies to this topic

#1 JackieG

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Posted 14 May 2010 - 07:25 AM

Hi,

Using regular primers on our samples resulted in products, however, now that we are using the same primer set with fluorescent dyes (Fam and Hex) we don't get any amplification. Are there any obvious changes to our PCR cycle that we should be making?


Thanks.

#2 chrisbelle

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Posted 18 May 2010 - 11:54 PM

Hi,

Using regular primers on our samples resulted in products, however, now that we are using the same primer set with fluorescent dyes (Fam and Hex) we don't get any amplification. Are there any obvious changes to our PCR cycle that we should be making?


Thanks.


Do you mean the primers are internally labeled with FAM and HEX? Or do you mean 5' nuclease assays?
Life sucks. Enjoy it while you can.

#3 Micro

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Posted 30 May 2010 - 10:58 PM

I'm having the same problem. When I use my HEX labelled primer (for the PCR product to be used for T-RFLP) it doesn't work under the conditions that normal primers do.

To answer Chrisbelle's question: in my case it is the HEX is at the 5' end.

Any suggestions on what could be causing this problem?

Cheers
M :)

#4 Micro

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Posted 17 June 2010 - 05:03 PM

Just in case someone comes looking for help on this topic in the future..... I spoke with the company that manufactured my primers. They indicated that although their supplier of the marker does not indicate that there should be problems, in their experience some primers which have dye such as HEX, FAM and JOE on occassion just don't work. They suggested first optimisation of the PCR condidtions for the labelled primers. If this doesn't work, they said some people have has success by moving the primer up or down stream of the current primer target site or changing the marker to opposite primer (i.e. marker on forward, then you would change it to reverse). Cheers M :P

#5 Maddie

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Posted 21 June 2010 - 11:43 AM

Thanks for the info. Interesting.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein




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