different amount of template DNA added will give out different pattern in gel af - (Apr/03/2006 )
Please help~~~~
I am doing a project which is about bacterial identification by AP-PCR. I have chosen 4 different amount of template DNA added (10ng, 15ng, 20ng and 30ng) for running PCR. The purpose of the step is to find out how many DNA added can give out the best pattern. After gel electrophoresis, I found that the reactions (10ng, 20ng and 30ng DNA added) gave out less number of bands. However, the reaction (15ng DNA added) had more bands. The extra bands had smaller molecular weight. I would like to know why adding different amount of template DNA which will give out different pattern in gel?????
Sorry, my English is not good...Please forgive me ! Thanks !!!
Differing the amount of template in the reaction may increase the 'amplifiable' copy number of each targeted sequence. Adding too much template can limit the reaction. Depending on the template you can have a bell shaped curve of results, with the best results at the middle concentrations and less reliable results at the low and high concentrations.
Smaller sequences will amplify more readily in PCR reactions, due to the kinetics of the process. If you have a mixture of template sizes or targets, smaller targets (bands) will amplify more quickly than larger targets, take over the rxn, reagents and be more visible on a gel.
Smaller sequences will amplify more readily in PCR reactions, due to the kinetics of the process. If you have a mixture of template sizes or targets, smaller targets (bands) will amplify more quickly than larger targets, take over the rxn, reagents and be more visible on a gel.
Thank you so much !
I still have a question. You said that adding too much template can limit the reaction. The more the 'amplifiable' copy number of each targeted sequence, the higher chance of amplification. Isn't it? How to limit the reaction?!
I'm not exactly sure how it 'limits', but I do know too much template will result in a smeared PCR product (on a gel). 'Limits' may not be the right term. I suspect too much template saturates your rxn components in the initial cycles of the pcr.
Anyone have any suggestions about the specifics here...?
I think smears after PCR, caused by overabundance of template (i.e., they go away when you dilute your template 
) may in part be due to non-specific primer-binding. even if the interaction is inefficient, if you have whopping amounts of gDNA in there, some primers are gonna try to bind in places where the affinity is not so great, leading to amplification of many different sizes
but it's only a theory, I haven't tested it or anything! 