i've got a problem with the pcr select cdna subtraction kit from clontech. everytime i made the pcr (V.c. in the manual) to control my adaptor ligation efficiency i got a smear from 0,1kb to 2kb instead of a diskret band at 1,2kb. this was made with GAPDH3' primer and PCR primer 1, which binds to the adaptor. my internal control made with GAPDH3' and GAPDH5' primer shows a correct band at 0,5kb.
can somebody tell me why i always get a smear instead of a discret band?
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pcr select cdna subtraction
Started by hotscho, May 03 2002 04:36 AM
1 reply to this topic
#2
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Posted 27 May 2004 - 12:00 AM
Hi
probably its no loger relevant but its time someone responded!
it sounds like your GAPDH3 primer may not be specific under the PCR conditions you are using, so that you get amplification of the entire library. the GAPDH5 primer may be more specific (higher tm perhaps?) so when you them two no problem. I would try repeating with lower annealing temperaatures.
Good Luck
probably its no loger relevant but its time someone responded!
it sounds like your GAPDH3 primer may not be specific under the PCR conditions you are using, so that you get amplification of the entire library. the GAPDH5 primer may be more specific (higher tm perhaps?) so when you them two no problem. I would try repeating with lower annealing temperaatures.
Good Luck
