No PCR Product - Fungal Genomic PCR (Nov/21/2005 )

I had focus on ampilifing my fungal dna for months, till now i still don't have any bands in gel. I use the universal primers ITS1 and ITS4 for the 18s rDNA and I brought Platinum taq from invitrogen recently. I follow the recommended recipe preparing the master mix, still there is no result from the pcr.

The condition I used last time is this:
initial 95 deg 10 min
35cycles 94 deg 30 min
45 deg 30 sec
72 deg 1 min
last 72 deg 10 min

I'm totally frustrated on this,pls help me>_<

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If that was not a typo, you've killed your Taq at the first "94 degree for 30 minutes" step... Also, your 95 degrees for 10 minute initial step seems a little excessive...

Try:

1. 94°C 2 minutes
2. 94°C 30 seconds
3. 45°C 30 seconds
4. 72°C 1 minute (or 1 minute per kb)
5. 72°C 5 minutes
6. 4°C hold

Run steps 2-4 for 35 cycles. BTW, 45°C seems awful low for an annealing temp, and if you're using high-fidelity Taq from Invitrogen, lower the extension temp to 68°C.

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thx for your advice
I will have a try

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HomeBrew,

I followed your suggested condition for pcr and ran gel
still, there is no product ampilfied.............TT^TT
whatelse I can do now

Thx

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Asumming your primers are good (are the "ITS1 and ITS4 universal primers" well known?), I'd try Invitrogen's PCRx Enhancer kit next.

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Unless these are degenerate primers I would definitely try a higher annealing temperature. In my experience, 45C annealing essentially never works. Try a range from 52-60C. This is most easily done with a gradient cycler, but if you don't have one, then try 55, 52, 58 in that order. Perhaps you could load the primer sequences and we could look at them.

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phage434,

I did a gradient pcr with annealing temperature from 51 to 65 degree
again...no result..........sign

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Googling for "ITS1 ITS4" leads to this article which uses a 58C annealing temperature.
You should look carefully at this article, as well as its reference 47, the original source of the ITS1 ITS4 primer pair.

Journal of Clinical Microbiology, August 2002, p. 2860-2865, Vol. 40, No. 8
Rapid Identification of Pathogenic Fungi Directly from Cultures by Using Multiplex PCR
Guizhen Luo and Thomas G. Mitchell

47. White, T. J., T. D. Bruns, S. B. Lee, and J. W. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, p. 315-322. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols. A guide to methods and applications. Academic Press, San Diego, Calif.

I'd guess that your genomic DNA prep has problems... Perhaps contaminating phenol?
Have you gotten any control PCR reactions working? Have you thought about making your primers again -- they are cheap, your time is not.

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phage434,

I use qiagen dneast plant mini kit to do the extraction
I assume the product won't be that worst........

thx for your reference, i will take a look at them

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For Fusarium genomic dna I couldn't get PCR products until I increased the initial denature step to 95 degrees for 5 min and the cycling denature temp to 95 degrees also. Try 95 deg for 5-10 min and then cycle with 95 deg for 45 sec.

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