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PCR product running on agarose gel - (Feb/20/2013 )

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Though theoritically a single DNA molecule is sufficient to amplify a fragment through PCR, practically its not possible as this single molecule has to be intact until it reaches the polymerization phase of PCR. so upto me 6ng of genomic DNA you are using/reaction is not enough. i would suggest a MINIMUM of 30ng of genomic DNA is needed for a PCR. mostly people use 100ng of genomic DNA. since your DNA is extracted form faeces make your your DNA is intact (by running agarose gel) and spec read its purity. so scale up your DNA concentration accordingly and check.
now what i think is the dimers you are seeing might not only because they are more prone to anneal themselves, but might also be due to insufficient template in the reaction.

good luck...

-GNANA-

Thank you so much for your suggestions GNANA, I really appreciate it.

-vanu-

Im sorry to disturb you again but I have just one last question, regarding the amount of template. In my extractions, the maxium concentration I obtained is 100 ng/microlitre more or less, and if I would like to add 30 ng to my reaction, I would need a stock of 375ng/microlitre, which I havent been able to obtain...is there a way to increase that concentration, I mean, woud I be able to sum the solutions obtained in different extractions (it may be a stupid question...)

-vanu-

vanu on Sun Feb 24 17:54:30 2013 said:


I´m sorry to disturb you again but I have just one last question, regarding the amount of template. In my extractions, the maxium concentration I obtained is 100 ng/microlitre more or less, and if I would like to add 30 ng to my reaction, I would need a stock of 375ng/microlitre, which I haven´t been able to obtain...is there a way to increase that concentration, I mean, woud I be able to sum the solutions obtained in different extractions (it may be a stupid question...)

But then you confuse the volume of the mastermix (10-50 microlitre) and of your DNA sample.(x microlitre)
You want 30 ng DNA in the mastermix and for this you have to add 0.3 microlitre of the DNA sample for each reaction.

-hobglobin-

I see what you mean and Im a little bit confused.

The calculations that Im doing are:
C1=My stock: 100ng/ul
V1=Vol that I add from the stock to the reaction: 2 ul
V2=Final volumen of my reaction: 50 ul
C2 The concentration of my template in the reaction?
Taking into account C1. V1= C2. V2

100. 2= C2. 50
C2= 4 ng/ul, so this is the concentration of my DNA in the PCR reaction.

Is that right?So, if the pfu polimerase protocol says that the DNA template maximun concentration should be lower than 0.5 ug/50 ul is the same as 10 ng/ul, isnt it?
So to obtain C2=10 ng/ul I would need a stock of...
C1. 2= 10.50
C1=250 ng/ul and thiis is my problem that I cant have this concentration from where to start...

I might be mixing quantities and concentration concepts...thank you for your patience and help

-vanu-

But 4 ng/microlitre is less than 0.5 ug/50 ul (= 0.01 ug/microlitre = 10 ng/microlitre).
Anyway I'd go with DNA as absolute value that should be in the mastermix that is 10-100 ng genomic DNA, and don't calculate the final concentration.

-hobglobin-

yes I know that, but if I want to add DNA at 10 ng/ul, I need the stock to be at 250ng/ul, and as I cant extract that large concentration, I was asking if there is a way to increase my stock concentration by making different extractions

-vanu-

vanu on Sun Feb 24 19:12:29 2013 said:


yes I know that, but if I want to add DNA at 10 ng/ul, I need the stock to be at 250ng/ul, and as I cant extract that large concentration, I was asking if there is a way to increase my stock concentration by making different extractions

okay then you have 500 ng in a 50 microlitre reaction which is far beyond any recommendation, anyway for this you can use columns (kit) or make an ethanol precipitation and resuspend the DNA in a smaller volume finally

-hobglobin-

see vanu dnt get confuse with concentration and obsolute value. let us just go with wat your pfu protocol says, as u mentioned they say a maximum of .5microgram/50 microlitres (obviously this s far beyond any recomendation as hodglobin said above), which means you will be having 500ng DNA in a PCR reaction. even if you want to use the maximum conc as your protocol says, your template stock having now is enough concentrated and no need to concentrate it further, that is your conc now is 100ng/microlitre, in which if you add 5 microlitres, the total DNA you have added now is 500ng or 0.5 microgram/50 microlitres or in your words it is 10ng/microlitre (for 50 microlitre PCR reaction). but this is way too much,
so if you do PCR with a TOTAL of 30 ng and above upto 100 ng/reaction or even 200ng/reaction, you got to add 0.3 microlitres (30ng) to 2 microlitres (200ng) from your current stock (100ng/microlitre).

-GNANA-

Fecal material often (always?) has strong PCR inhibitors. I'd suggest the opposite of most of these suggestions -- that you dilute your sample, perhaps 100x. I'd also strongly recommend that you attempt to find a positive control of some sort to check your primers and verify the appropriate annealing temperatures. Are you using a master mix? I'd suggest one. While mixing can provide more flexibility, that is often a problem not a feature, especially if you have no amplification, as in this example. If you continue to have problems, please report the exact primers, intended template, enzymes, and cycling conditions you are using and we can perhaps help more.

-phage434-
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