Hi Henry

The problem (smear from well to bottom of the gel) which is seen in your gel is mostly observed when your template is significantly more than required, or also if more enzyme units are added or ..if Mg or any other metal ion is in excess.. or if you do PCR for more number of cycles than actually required.........or if you use lower annealing temperature that is suitable for a clean fragment

.As you have used PAGE purified fragment..you should optimize the amont of template to be used for a clean DNA band without any smear...In case of you Taq (chinese source..2nd lane) there seems a correct sized product but accompanied by lot of smear.....you need to decrease template or PCR cycles etc.....But still puzzling is why another enzyme should give smear....As many of the factors are mentioned above by Ananda...check for the units of Ex-Taq form Takara you are using,,,check the buffer composition and try to optimize it.....

.But I suggest..you decrease some amount of template.....decrease number of cycles....and also the time of extension in the PCR (don't give unnecessary long extension times).....IT IS A POSITIVE SIGN THAT YOU ARE GETTING THE PRODUCT WITH ONE ENZYME...SO TO GET WITH ANOTHER ENZYME...SOME OPTIMIZATION MAY BE REQUIRED

..

.AND ALSO..............CERTAIN RECOMBINANT PCR AMPLIFICATION ENZYMES HAVE MODIFICATIONS/MUTATIONS TO ENABLE THEM TO BIND THE DNA MORE STRONGLY FOR HIGHER PROCESSIVITY....THIS MAY ALSO AFFECT THE STRINGENCY OF PRIMER:TEMPLATE INTERACTIONS.....SO OPTIMIZATION FOR ANNEALING MAY ALSO BE REQUIRED.....CHECK THE LITERATURE FOR EX-TAQ FROM TAKARA CAREFULLY....

all the best

I have got similar problem of getting the whole sample on the well while amplifying large DNA fragment, but I wonder in your case, differnt enzyme can modify the result!?! May I ask you the size of your DNA fragment?

The primer:

s:(5' gatttgttttttgtttggattg 3' )as: 5'( ataacctaatccaaacttatttccc 3')

the aimed dna fragment :

gatttgttttttgtttggattggagttttgttggatttaggaggtttttatattttatttttaggttatgtatattttagttggcgtaggacgttttttatgggtcgttttattaaatatatgtgtatttgggataataaaagattagtattattgtaaatatataattgagtttaaaggtagatatcgtttttttttttttttgtttttaagacgaggttaggaatagggaaataagtttggattaggttat

Ok to my knowledge, I have no special answer, but what I would do is, I shall make an other control reaction with the enzyme Ex-Taq(which you like to use?) to amplify your specific gene and an other well known gene(around 500-1000bases) from any other construct for example. So you can make sure that the enzyme is still functional (and not it is contaminated or may be some other hindrance making this! like any buffer,salt,or sometime the gel itself who knows). In addition, you can also try any other high fidelity enz such as Pfu polymerase etc..

Good luck!

Hi,

I guess it's mispriming along with correct target amplification. You can just increase the annealing temperature to get rid of unspecific band.

Every thing else looks ok.

Hi Henry

The problem (smear from well to bottom of the gel) which is seen in your gel is mostly observed when your template is significantly more than required, or also if more enzyme units are added or ..if Mg or any other metal ion is in excess.. or if you do PCR for more number of cycles than actually required.........or if you use lower annealing temperature that is suitable for a clean fragment

.As you have used PAGE purified fragment..you should optimize the amont of template to be used for a clean DNA band without any smear...In case of you Taq (chinese source..2nd lane) there seems a correct sized product but accompanied by lot of smear.....you need to decrease template or PCR cycles etc.....But still puzzling is why another enzyme should give smear....As many of the factors are mentioned above by Ananda...check for the units of Ex-Taq form Takara you are using,,,check the buffer composition and try to optimize it.....

.But I suggest..you decrease some amount of template.....decrease number of cycles....and also the time of extension in the PCR (don't give unnecessary long extension times).....IT IS A POSITIVE SIGN THAT YOU ARE GETTING THE PRODUCT WITH ONE ENZYME...SO TO GET WITH ANOTHER ENZYME...SOME OPTIMIZATION MAY BE REQUIRED

..

.AND ALSO..............CERTAIN RECOMBINANT PCR AMPLIFICATION ENZYMES HAVE MODIFICATIONS/MUTATIONS TO ENABLE THEM TO BIND THE DNA MORE STRONGLY FOR HIGHER PROCESSIVITY....THIS MAY ALSO AFFECT THE STRINGENCY OF PRIMER:TEMPLATE INTERACTIONS.....SO OPTIMIZATION FOR ANNEALING MAY ALSO BE REQUIRED.....CHECK THE LITERATURE FOR EX-TAQ FROM TAKARA CAREFULLY....

all the best

Thanks a lot for help. I usually use Ex-Taq enzyme to amplify my DNA template. The Taq enzyme is just used for the PCR whose template is bacterium fluid . The most strange part is that I did get some right sequence by applying Ex-Taq with nearly the same template and same PCR system occasionally. Maybe the buffer of EX-taq or excess metal affected my result, however, the PCR reagents were brought from company, there is no way to change the certain amount of one or two parts of the buffer. what I can do is just to change the whole amount of buffer in PCR system. And the cycle times ,annealling temperature, were all have been considered during many times of the same experiments.

What I guess from this picture is that you have loaded more than required gel. I would do the following, if I would be sure of my amplification. The firs thing is: either your DNA in the first well is not amplified as it has to or there might have been issue of mispriming as suggest by some of our friends in earlier replies. I would recommend you to load the less amount of DNA and make sure your Gel is of accurate concentration as suggested in protocol. If that is true, try amplifying your first well DNA with the conditions suggested in the protocol.

In times that this has happened to me I diluted my template (1:10 and 1:100) and changed the gel components. Once my TAE had gone bad and once my agarose was somehow affecting the outcome. I'm not familiar with the types of Taq you mentioned, but perhaps double check to make sure it doesn't require a 'hot start' cycle. Although you probably wouldn't be getting anything if that was the problem. . .

Another probable reason of getting these kind of bands may be that you are using very high voltage from your power pack which is actually not required for your sample.