PCR improvement - how to get more PCR product! (Apr/02/2008 )

hallo,

i want to improve my pcr to get more specific product for gelextraction. first pcr worked well (1 product with right size), then i tried to change some parameters (DNA amount, cycle number) and what i got was many bands.

to show i attached picture.

what can i do to get more product?

thanks for suggestions

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40 cycles is way too much! You're amplifying up other crap (hence why you have so many bands). Try using more template and decrease the number of cycles.

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Haha, I'm pretty sure they K.Mullis was once quoted as saying in some many words, "You're an idiot if you have to use more than 30 cycles to amplify DNA." So yeah, I always use 30 cycles. If you're amplifying from a plasmid, 10ng of input is more than enough, genomic you'll want more like 100ng. The best thing to optimize to enhance product yield for me has been messing with the annealing temp. Get a gradient cycler and figure out the best Tm.

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ok. your right. 30-35cycles are enough.
i don´t use plasmid (the it would be easy), and no genomic DNA.

i use cDNA. and tried annealing temp. 58°C (see picture 1, one nice product!!) with 60°C no product!!

how can i improve amount of product? can i retry procedure with same annealing temp (58°C) and more template?

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Different primers, or more likely in this case, nested primers, using your first result as template for the nested primers. Use very very small amounts of the first reaction as template for the second.

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This is how I try to solve such problems:

try a touchdown PCR (I don't know if this is useful for cDNA, but with complex samples the results sometimes are fantastic)


try a temperature gradient and different Mg++ concentrations and different template concentrations (if this is not sucessful vary every parameter in the PCR mix you can think of!)


clean the PCR product to remove primer dimers (cut out your band or use spin columns or PEG) and reamlify your sample


hope this helps!

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For a start, I think your primer Tms are too far apart. If you reduce the Tm of the forward primer (remove the 3' AC dinucleotide) and/or if you can add a couple of bases to the reverse primer, your Tms will be much closer together, and your specificity will increase, because you can anneal at a higher temp.
Next, if your Tms are about 75C, I would anneal at 60-65C, and extend at 72. Better still, as was suggested, try a touchdown PCR. I'd start the touchdown from 5C less than the Tm of the higher primer.
Finally, 30 cycles should be enough to get tons of product.

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