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Troubleshooting methylation primers for Bio-Rad PCR


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#1 Moemedawar

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Posted 24 November 2013 - 11:35 AM

I was told that bio rad PCR has some issues with methylation primers where incompatible primers could break the DNA strands and thus no amplification takes place. The solution is to pick compatible primers based on certain guidelines developed for this issue. Does anyone know how this can be solved? Or where I can find these guidelines?
Thank you

#2 pcrman

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Posted 10 December 2013 - 10:15 PM

What do you mean by "bio rad PCR" - their PCR machine or reagent? I don't understand how " incompatible primers could break the DNA strands". Can you clarify?



#3 Moemedawar

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Posted 11 December 2013 - 11:42 AM

What do you mean by "bio rad PCR" - their PCR machine or reagent? I don't understand how " incompatible primers could break the DNA strands". Can you clarify?

Hello pcrman,

 

I was referring to the BIORAD PCR machine. Regarding the primers, I was told by one of the faculty at my university that I need to pay close attention to the constraints laid out when using BIORAD machine when doing Bisulphite specific PCR, and that there's a protocol or a list of specifications the primers have to have in order for the primers to be compatible with the machine. I don't know exactly what the nature of the constraints is or why they occur in this machine and not others, but what I was told is that if I choose incompatible primers, I will not get an amplification product because of the reason I mentioned earlier.

 

Any clue?



#4 Ameya P

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Posted 13 December 2013 - 12:50 AM

Probably the faculty meant that Methylation specific Primers need to be designed with much care, otherwise you would not get any amplification. 

 

I am no expert at PCR but in my opinion, Primer and Cycler incompatibility should not one of the reasons to cite for PCR failure. 


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