Lack of repeatability in RT PCR

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allurivenkatesh
allurivenkatesh's picture
Lack of repeatability in RT PCR

I am using Invitrogen super script two step kit  . i have problem in repeatability in RTPCR . I get required size band in pilot reaction with good intensity , if i repeat the same RT PCR taking the same RT product as template in more copies to elute the amplicon , i dont get the same good intensity band that i get in pilot reaction . This is happening repeatedly . How to solve this lack of repeatability in RT PCR .
                                              

Waiting for quick reply  ,
Thanking ypu ,
Venkatesh Alluri

Ivan Delgado
Ivan Delgado's picture
 

 
Typically if you are getting good amplification the first time and then see the intensity of your band drop on subsequent reactions, you may have an issue with template stability. If you are freezing and thawing your RT product, then you are likely breaking the cDNA down and as a result you have less template for your PCR. Your best bet if this is the case is to aliquot your cDNA into different tubes and only thaw your template once you are going to use it. Of course this assumes that you are storing your cDNA at -20oC and that it never thaws in the process (some -20oC freezers defrost automatically, something that would be very bad for your cDNA). 

Good luck

allurivenkatesh
allurivenkatesh's picture
Tanq very much i will

Tanq very much i will definitely work on it. i will let u know the result of ur suggestion .