Smearing of Positive DNA Samples (NOW with JPEG!) - (Nov/17/2005 )

Hi

I have recntly started using the Taq Polymerase by Promega and I make my TaqMix by adding dNTPs, reaction buffer and the Taq polymerase enzyme. I used to use "Taq and Go" by Qbiogene. I have recently ran several PCRS and on my expected posiive samples I had smears for the length of the hyperladder. This is regardless of whether I use 250nM or 10uM primer concentrations. The negative lanes stay nehgative so I dont think it is mass contamination. Any ideas anyone as since I have changed Taq I am going backwards!! The primers were for Lactobacilli sp. Please see TIFF image to see what I mean. Lane 1 is hyperladder, Lanes 2 and 3 are L. casei and L. acidophilus, Lane 3 is E.coli and Lane 4 is control (MG water) using 250nm of each primer.
Lanes 6 and 7 are L. casei and L. acidophilus, Lane 8 is E.coli and Lane 9 is control (MG water) using 10um of each primer.

Thanks,

Is it possible that what we are seeing is your template??? Maybe there is not any amplification

I cannot speak to the exact products you have used, but each company's kit is different, perhaps a different mg++ concentration in one kit than the other means your primers work with one kit and not the other??

As to primer concentration, we usually use 0.5uM but I would play with Mg++ concentration before adjusting the amount of primer...

One last comment, I like rTaq from Takara, it has been a good (high efficiency) and consistent enzyme for me, at first glance it looks pricey, but each tube comes with a new stock of dNTPs... Anyway I am not their salesman, but I do like this product...

HTH

Have you run PCR reactions with dilutions of your template? A smear is sometimes indicative of too much template. If there is too much DNA in the reaction tube, primers get soaked up by the template in an non-specific manner resulting in templats of all different sizes hence the smear.

quote name='JamesKnowles' date='Nov 18 2005, 09:35 AM' post='31639']
Hi

I have recntly started using the Taq Polymerase by Promega and I make my TaqMix by adding dNTPs, reaction buffer and the Taq polymerase enzyme. I used to use "Taq and Go" by Qbiogene. I have recently ran several PCRS and on my expected posiive samples I had smears for the length of the hyperladder. This is regardless of whether I use 250nM or 10uM primer concentrations. The negative lanes stay nehgative so I dont think it is mass contamination. Any ideas anyone as since I have changed Taq I am going backwards!! The primers were for Lactobacilli sp. Please see TIFF image to see what I mean. Lane 1 is hyperladder, Lanes 2 and 3 are L. casei and L. acidophilus, Lane 3 is E.coli and Lane 4 is control (MG water) using 250nm of each primer.
Lanes 6 and 7 are L. casei and L. acidophilus, Lane 8 is E.coli and Lane 9 is control (MG water) using 10um of each primer.

Thanks,[attachment=310:attachment]
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Could this be a problem of primer design..if only one of the primer is priming good and the other one is not very good a primer could u have a smear???...I dont mean to confuse you..but one check to do is to see if the designed primers are priming right..