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-   -   Single primer giving spurious band at the desired position after 3' RACE .. (http://www.molecularstation.com/forum/pcr-polymerase-chain-reaction-forum/70541-single-primer-giving-spurious-band-desired-position-after-3-race.html)

ari 09-08-2009 07:58 AM

Single primer giving spurious band at the desired position after 3' RACE ..
 
Hiiii everybody....

I am a new member to this community and this oneís my first post.. actually i'm facing some problem in doing 3' RACE.. though i've designed the Forward Primers maintaining stringent parameters and have designed from the ROH of other organisms from the same group... when i performed the 3' RACE using the cDNA (synthesized using cDNA synthesis kit from Fermentas, in case of 3í RACE I didnít use any kit) in some cases i found band at the desired region along with various other multiple bands at different positions.. But to crosscheck it when i performed Negative Control PCR using just the Forward Primer in one reaction and Reverse Primer i.e. the Anchor primer in another reaction (using double conc. of the primer in each case) resp., to my surprise i got the band at the same desired region in one of the two reactions which i got when i did 3' RACE... tried with different primers but facing the same problem... i am really confused and dont know how to solve this kind of problem when the single primer is giving away the band at the desired position... Can anybody please suggest how to overcome such problem and a way to distinguish the desired band from those spurious bands at the same region... ???.........


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