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Rt-pcr

Rt-pcr - PCR - Polymerase Chain Reaction Forum

Rt-pcr - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 06-02-2008, 10:53 AM
Pipette Filler
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Default Rt-pcr



rtpcr

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while doing rt pcr when i am using 20pM conc. of primer
i did not get band in -ve ctrl,
i got band in +ve ctrl
and light band in test.
good results
but.....
in order to increase the darkness of band in test i increase the conc of primer to 40pM and 100pM now i am getting more than two bands in ctrl as well as in test

can any one tell me whats the reason behind it
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  #2  
Old 07-08-2008, 06:51 PM
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Default Re: Rt-pcr

probably unspecific products due to too much primers. don't change your primer concentration. if you want a darker band simply increasing the number of cycles would do it. 5 more cycles are usually more than enough. too many cycles and unspecific products might start to appear again.
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Old 07-09-2008, 09:43 AM
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Default Re: Rt-pcr

thanks i will try then tell.
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Old 08-30-2008, 01:28 AM
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Default Re: Rt-pcr

may be this due to increase the primer conc.
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