Can primers go bad and give a smear in PCR? - (Jan/27/2007 )
QUOTE (aimikins @ Feb 2 2007, 03:13 PM)
why don't you just order a fresh set of primers? it's a cheap fix...and sure they can go bad, it happened to me once a while back and it took me a while to figure out that it was the cause of my problems
Hello
I have the same problem as Clementine: a primer that stopped working progressively (first it gave dimers along with the correct band, then weak band, then just smear looking like clementine gel pic) while other primers work well.
so I order a fresh set of primer but this one didn't work from the start!
My product is supposed to be 220 bp. I use an annealing temperature of 50 and I tried to lower it to 45 with longer extension time but still got the same bad results while it worked previously. I even tried on different thermal cyclers.
Any explanation?
Thanks
-eye am-
annealing temp at 50 is very low... i suppose it's raising unspecificity which may be translated in smearing on gel.
-fred_33-
QUOTE (eye am @ Sep 15 2007, 01:27 AM)
QUOTE (aimikins @ Feb 2 2007, 03:13 PM)
why don't you just order a fresh set of primers? it's a cheap fix...and sure they can go bad, it happened to me once a while back and it took me a while to figure out that it was the cause of my problems
Hello
I have the same problem as Clementine: a primer that stopped working progressively (first it gave dimers along with the correct band, then weak band, then just smear looking like clementine gel pic) while other primers work well.
so I order a fresh set of primer but this one didn't work from the start!
My product is supposed to be 220 bp. I use an annealing temperature of 50 and I tried to lower it to 45 with longer extension time but still got the same bad results while it worked previously. I even tried on different thermal cyclers.
Any explanation?
Thanks
What are the theoretical Tms of the two primers. Ideally, you should have them within 4-6 degrees of each other, ust to make things simple for yourself.
If the Tms are close, try a touchdown PCR. Begin with an annealing temp a couple of degrees above the theoretical Tm of the higher-Tm primer (you do know that calculating Tm is a bit of a 'black box', don't you?), then after 4 cycles drop it 1 degree. Every 2 cycles after that, drop another degree until you are about 5 degrees below the theoretical Tm of the lower primer. This will ensure that the correct templates will have an advantage over the misprimed templates.
also, check your Mg concentration.
-swanny-