PCR troubleshooting - (Dec/17/2008 )

Could you try to clean your PCR product with a clean up kit and then use it for further PCR reactions? Using the PCR product directly (without clean up) could possibly interfere in your second reaction and as a result you don't get a product.

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The clean up kit you have said means the GEL PURIFICATION KIT?I didnt do the gel purification because the first pcr result buffer is 20 microliter about.It is so few that I want to reamplification it through the second PCR.I cant repeat the same result as the result of the fisrt PCR.

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how big is your DNA band?

there are gel purification kits and PCR purification kits. Do you have a PCR purification kit?

If not you can use gel purification kit in place of a PCR purification kit so long as your PCR product is over 100bp. (Just imagine that the PCR solution is a gel). Both kits work on the same principle except that the silicon column is optimised to bind different size range of DNA fragments.

I would prefer diluting a sample of the DNA before running the second PCR. DNA polymerase is inhibited by pyrophosphate, a byproduct of the PCR reaction.

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If you still have the tube which had your first successful pcr product, you can probably amplify from it, even though you think it is empty. Add 10 ul of water and use 1 ul as template in a new reaction. You likely were using far too much of the first product as a template, or have some more fundamental problem.

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There is 2 microliters left in the tube of the first PCR product.I diluted it for 10,25,100 times as the template of the second PCR again. NO result band can be obtained too.

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What about redoing the PCR ? Not feasible?

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The attached picture is the repeat PCR with the genome as template.
M=mareker,100,250,500,750,1000,2000,3000,5000
A1,B1,C1:The first PCR with the genome as template.
A2,B2,C2:The second PCR with the first PCR product as template.(add 5 microliters of the first PCR product to the second 25 microliters PCR buffer directly)
A,B and C has the different primers concentrations.A has 20 times more primers than B,50 times than C.

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It seems like your PCR is not working this time around unfortunately. and it seems like A2,B2,and C2 might be using the genome template not A1,b1,C1. And how much exactlyu genomic DNA u are using?

Mastermix PCR or manually mix?

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I had got the same result picture with the different primers to amplify the same gene.I didnt annotate wrong about the picture.
I use probably 0.2ug genomic DNA in 25 PCR reaction buffer and use 2 * masermix PCR(3mmol/L MgCl2,0.2mmol/L dNTP,0.1U/ul Taq DNA polymerase)

I dont know why the simple principle PCR has beat me so heavily.

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I agree with this suggestion. You should be able to get product from this. I would try lowering the annealing temperature all the way down (near 37) to see if you get any product (non specific or otherwise) to check that your PCR reaction mixtures are working. Alternatively, include a positive control.

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