Can primers go bad and give a smear in PCR? - (Jan/27/2007 )
Hi to everyone,
I am having difficulties in some of my PCR reactions.
There was a set up protocol already for these primers in our lab and first time I did it, it worked perfect.
Afterwards, I could not get any successful result.
All I get is smear which starts even from the lanes.
I tried another primer pair with the same reagents and this does work.
I am wondering if the primers can go bad and give such a smear. If so how??
Thanks for the replies
yes, primers can go bad.
However this happens very very rarely. Primers go bad when subjected to repeated freeze-thaw cycles and repeated use. (which increases the probability of contamination from nucleases and bacteria.. most of which come from human skin and saliva - produced as a fine aerosol whenever anyone speaks)
However as mentioned, primers very rarely go bad... because there are so many primer molecules, all one experiences is a drop in product yields rather then absolute failure.
The more likely cause of problems is template degredation. Please cheack your template to make sure it hasn't started degrading.
Will it be the agarose gel or perhaps the problem is in the used TAE buffer?
For primer, I think it is always a good idea to aliquot some out and store the others to reduce thawing effect.
It is possible for primers to degrade. But I have never had one degrade, thats why its not the first thing to suspect.
Was the second PCR with the same template ?
would you mind posting a pic of your results, please?
as many person that uses these primer set the more that they will go bad. Each person should prepare a working solution for own use and let a stock soln. so the primers will mantein good for longer. for pcr is better to use filter tips. when thaw let them in ice don't force the thawing by placing in the hand and always mix by invertion of tube, the vortex uses to much force.
Dear All,
Thanks for the replies.
Here I attach my PCR picture.
Initially, I made PCR with this primer pair and it always gave smear. I tried to change evrything but it still continued (The DNA template is sonicated genomic DNA).
Finally I used a different primer pair (indicated as mIP10 in the picture) to amplify from this same template and there was a clear nice band at the expected size and the CTRL which does not have template in it, did not give a band (as expected).
This is why I think it is primers which give smear but I can not explain how!
I would be glad to hear your comments.
Thanks
Dear All,
Thanks for the replies.
Here I attach my PCR picture.
Initially, I made PCR with this primer pair and it always gave smear. I tried to change evrything but it still continued (The DNA template is sonicated genomic DNA).
Finally I used a different primer pair (indicated as mIP10 in the picture) to amplify from this same template and there was a clear nice band at the expected size and the CTRL which does not have template in it, did not give a band (as expected).
This is why I think it is primers which give smear but I can not explain how!
I would be glad to hear your comments.
Thanks
everything is the same? water, dNTP etc.....try same wells on the PCR cycler. after all your primers might be contaminated.
are the primers that worked for the same portion of sequence as the primers that don't work?
maybe your genomic dna is sheared within the sequence of your primer pair.
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