Rt-pcr

[nadia naeem]

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

[halramin]

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.

[nadia naeem]

thanks i will try then tell.

[saly]

may be this due to increase the primer conc.