I want to get sharp bands of PCR products on the gel electrophoresis without any tailings, primer dimers or non-specific products. What is the best concentration of both primers and DNA to get this result. I know that there other factors like MgCl2 concentration, annealing temperature etc that affect the result but I need to know the best for DNA and primers.
Thanks in advance
Unfortunately there is no universal answer. The optimum primer and DNA concentration will depend on a number of factors - the sequence and length of the primers, variation in the sequence of the priming site, the quality of the template DNA, kind of template DNA (genomic, mitochondrial, plasmids...), etc. Even the type of tissue you're extracting (eg. coral or mollusc tissue can have a lot of polysaccharides that are difficult to get rid of). There are guideline amounts for particular templates which you can probably find in the literature that comes with your polymerase, but the range on those recommendations is generally very wide (e.g. for genomic DNA it's usually between 1ng and 1ug). In general, for clean amplifications you're better off using as little template as you can get away with - we often dilute our standard DNA extractions 1/100, or even as low as 1/10,000. Similarly with primers, the recommended concentration is generally between 0.05uM and 1uM, which is a huge range. Generally you're using a massive excess of primers, so if you have primer-dimers it may be worth while to reduce the concentration, but I've never had much success messing with the primer concentrations.
The only way to know what will work for your particular template and locus is to experiment. Good Luck.
Dear Mohamed ahmed Sebak
you can use PCR master mix from fermentas company which contain all the component needed in pcr in right amount and 1x of pcr master mix is used in each reaction . 50ng to 100ng dna concentration is enough
Unfortunately there is no universal answer. The optimum primer and DNA concentration will depend on a number of factors - the sequence and length of the primers, variation in the sequence of the priming site, the quality of the template DNA, kind of template DNA (genomic, mitochondrial, plasmids...), etc. Even the type of tissue you're extracting (eg. coral or mollusc tissue can have a lot of polysaccharides that are difficult to get rid of). There are guideline amounts for particular templates which you can probably find in the literature that comes with your polymerase, but the range on those recommendations is generally very wide (e.g. for genomic DNA it's usually between 1ng and 1ug). In general, for clean amplifications you're better off using as little template as you can get away with - we often dilute our standard DNA extractions 1/100, or even as low as 1/10,000. Similarly with primers, the recommended concentration is generally between 0.05uM and 1uM, which is a huge range. Generally you're using a massive excess of primers, so if you have primer-dimers it may be worth while to reduce the concentration, but I've never had much success messing with the primer concentrations.
The only way to know what will work for your particular template and locus is to experiment. Good Luck.
Additionally to Laura's excellent answer good primer design with one of the online programmes ( primer3 ) is essential for amplifying cleanly. An annealing temperature as high as will work properly for clean bands. Use of a hot start enzyme to minimise primer dimer. Also run the gel slowly and in the cool to minimise band broadening will make your bands look sharper as there will be less thermal diffusion of the post pcr dna
I agree with the above answers that DNA should be used in between 50-100 ng and primer concentration should be 0.25-0.4 uM.
Dear Mohamed,
I agree with the above comments. The gnomic DNA or primer concentration depend on the application (eg. sequencing) or species under study. Different Protocol applied within species or within applications.
Please find here is attached a pdf file from AB Applied Bio-systems.
Good luck.
totally agree with the previous answers, however I would like to add a small tip, that is unifying the DNA concentration before going to the PCR as it will give almost a perfect band especially if you are planning for subsequent digestion reaction. you can do that by run a dilution series of DNA samples including a non-diluted sample, and samples diluted at 1:10, 1:100, and 1:1000 etc. with ddH2O.
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