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Genomic DNA amplification


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4 replies to this topic

#1 arvind

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Posted 08 August 2004 - 11:46 PM

hi all

i tried to amplify a 1.6kb genomic DNA from a parasite genome. Though my primer is full length, should amplify 1.5 kb and my taq polymersae must amplify up to 5 kb, i cant able to get the amplification.
but i get an amplifiction at 480 bp.

can u suggest me something, im worried.

arvind

#2 InvisibleSurfer

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Posted 09 August 2004 - 03:19 AM

Hi,

If you give us more details of the time and temperature combinations you use, perhaps we could help you out. As a rule of thumb, and since the gene you want to amplify is quite big, remember to give enough time for the primers to find and bind to it and more importantly give more time to Taq so it can read along the full length of the gene.

#3 arvind

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Posted 10 August 2004 - 11:11 PM

Hi,

If you give us more details of the time and temperature combinations you use, perhaps we could help you out. As a rule of thumb, and since the gene you want to amplify is quite big, remember to give enough time for the primers to find and bind to it and more importantly give more time to Taq so it can read along the full length of the gene.

hi
i used denaturation at 95C for 5 min
95 1 min
55c annealing
35 cycles

#4 InvisibleSurfer

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Posted 11 August 2004 - 03:08 AM

This is not enough information, we need to see the temperatures and incubation times at each temperature, Tm of primers etc.

#5 SVTX

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Posted 20 August 2004 - 08:03 PM

Hi,

I have amplified upto 7 kb from human genomic DNA. If you are using high-fidelity Taq polymerase, try 94C-2 min, denaturation at 94 C - 20s, annealing temp-30s -1min, extension at 68 C- 2min. Do 30-35 cycles. For long-range PCR, using high-fidelity Taq, denaturation temp of 94 for 20s and extension at 68 instead of traditional 72 C are highly critical because of the depurination happening to the DNA at higher temperatures. My long-range PCR never gave the required band when I had used the traditional settings.
Good luck!
SV




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