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freaky primers - (Jun/12/2007 )

hi
i have a verys trange problem , i was working with a certain primer that a collegue of mine checked for sequence and told me that its ok it worked well for 2 PCR runs opn HUMAN DNA and suddenly it stopped working for no obvious reason so i started optimizing the conditions all over again and changing al;l chemicals used and changing the programme for annealing and the DNA SAMPLES but still no luck , so i ordered i new set of the primer and started working again and again it worked 3 runs then stopped !!!!!
i did everything , i changed wateer , other chemicals , used new ones and with different concentrations , changed the annwealing from way below the TM TO way above it , , i checked the sterlization , used DNA samples different , but still just a smaer and sometimes a very faint band inside the smear .... !!!! can anyone tell me wats that? glare.gif

-lula-

sounds to me like possible nuclease contamination?

-lauralee-

When the pimers stop running, check them on a gel. Sounds like nuclease or other from of primer degradation. If PCR runs once with your conditions it is not so likely that it is problem of cycle conditions or the other components of your mix.

-kr├╝melmonster-

i did check the primers on gel and found very very weak band !!!
but nuclease ? what dio u mean by that
and how come a new primer can be degraded twice so fast ???????

-lula-

a pipette might be dirty? Do you use filter tips?

Also aliquote your primers into a working primers and stock primers. The stock primers you have to guard with your life, the best treatment possible. So insituations of contamination you can dump the working primers and redilute the stock primers to make more working primers.

Anyway to verify that the primers are to blame? You can run a PCR on plasmid DNA? Or use two separate PCR reactions that uses that pairs a known good primers with one of the problem primers? Can you run a polyacrlamide gel on the primers to see if they are still intact?

-perneseblue-

i do make working and stock solutions of primers ,,,ad my pippets are ok

i dont have plasmid DNA ( how to gt it ?)

i didnt understand the part about trying the primers with good ones .....also do u think the type of taq buffer im using might be of importance ? i use NH4SO4 buffer should i use KCL buffer >??

-lula-