PCR components/DNA sample optimal amounts - ul of DNA and each component for a good reaction. (Aug/14/2008 )

Hi all
I am using the following amounts as ul per 25ul PCR reaction,but i do not get any products(no bands seen on gel after electrophoresis).So can anyone say what is wrong or what i can do to correct it.I feel the amount i take is not good.

here i go

5 x colourless gotaq flexi buffer 5ul
MgCl2 sol,25mM each 2.4ul
PCR nucleotide mix,10mM each 1ul
upstream primer 1ul
downstream primer 1ul
Go Taq DNA polymerase 0.5ul
template DNA 4.6ul(500ng genomic DNA)
nuclease free water 9.5ul
Total 25ul


Also what is the optimal DNA sample amount that is to be taken.In the above sample i did not dilute.

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Hmm... what is the source of your template DNA? And do you have the regular GoTaq buffer? It seems you are using the Flexi Buffer with regular GoTaq. Promega's site says that with regular GoTaq buffer the 1x Mg concentration is 1.5mM, so you could definitely try cutting down on the Mg your adding, but you aren't so far off that it should be killing the reaction.

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What are the cycle conditions and expected amplicon size? I use 50-100ng template.

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The DNA is genomic DNA from mouse tail (transgenic mice).I am using Promega GoTaq Flexi buffer and GoTaq DNA polymerase which comes as a package.

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The cycle goes as follows
95-2mts,x1
95-1mt,x35
58-1mt,x35
72-1 mt x35
72-5mtx1
4-infinite
the expected amplicon size is 600bp.Is using 500ng genomic DNA -a too much? I have tried with annealing temperatures of 55 and 62.i run the pCR on an Applied Biosystems instrument which asks for the cover temperature before starting the reaction and i usually key in as 95.
What or where should i try to change things.

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The function of PCR is to amplify from small amount.
So I would say the amount of DNA template is not a big problem.
Even if the DNA template is minimal, but with right condition and right primers, you should at least see something (a faint band of your product etc).
Maybe you should consider to optimize your PCR condition etc?

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yes i will optimize my PCR but with the above cycle conditions and the amplicon length of 600bp inaddition to annealing temperature,what else can be tried out.
I am trying to do a gradient PCR with Veriti AB instrument.
I find some protocols running as 2 step method without the final extension.Is this better than the 3-step method.

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As long as you are happy with your technique and you know your reagents work and you have tried a few different annealing temperatures and you are getting absolutely nothing at all then I would check/redesign primers. You could spend a couple of weeks trying DMSO, Betaine, Formamide, Mg conc which you probable cost you more time and money in reagents than getting new primers. If new primers fail than try KODXL polymerase, I have always found this to be excellent for gDNA.

good luck

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another thing is, what kind of organism's DNA you want to PCR from?
If it is a GC rich bacteria, it is normal if you face problem in PCR.
Redesign another pair of primer (with different primer sequence) is good idea too, since the price of a pair of primer is not that expensive.

My friend encounter a situation where the primer sent by manufacturer is spoiled (i don't know how). After she re-order (with same primer sequence), her problem solved.

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it is transgenic mice tail DNA and i am trying to do the genotyping(trying to optimize the pcr).Today i tried with fresh genomic DNA but the DNA concentration itself was very low to start with.it was only 40ng/ul.i tried to do the beta actin and found or think i saw a very faint(very,very,very) band in the region.
The primer sequence was given to me by the lab which was maintaining the transgenic mice colony,so i was confident it is the right one as it had workedd for them.

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