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How much DNA in PCR? - (May/24/2007 )

I got my A260 readings, which allowed me to figure out how much DNA I have in each of my samples.
What is the recommended amount of DNA in a single PCR reaction??

-BV82-

QUOTE (BV82 @ May 25 2007, 06:07 AM)
I got my A260 readings, which allowed me to figure out how much DNA I have in each of my samples.
What is the recommended amount of DNA in a single PCR reaction??


That depends on several things.
Do you have genomic DNA? Or plasmid DNA? For genomic DNA 0.5-1 mg, for plasmid DNA 1-2 ng is good to amplify a fragment by pcr.

-aspergillie-

QUOTE (aspergillie @ May 24 2007, 11:06 PM)
QUOTE (BV82 @ May 25 2007, 06:07 AM)
I got my A260 readings, which allowed me to figure out how much DNA I have in each of my samples.
What is the recommended amount of DNA in a single PCR reaction??


That depends on several things.
Do you have genomic DNA? Or plasmid DNA? For genomic DNA 0.5-1 mg, for plasmid DNA 1-2 ng is good to amplify a fragment by pcr.



I have genomic, fungal DNA.

-BV82-

QUOTE (BV82 @ May 25 2007, 02:06 PM)
I have genomic, fungal DNA.

Then I would add 1 mg DNA to the pcr mix.

-aspergillie-

QUOTE (aspergillie @ May 25 2007, 03:42 PM)
QUOTE (BV82 @ May 25 2007, 02:06 PM)
I have genomic, fungal DNA.

Then I would add 1 mg DNA to the pcr mix.


And I just saw your other question.
I've worked a lot with fungi, and pcr on fungal genomic DNA is not as simple as on other DNA. You could try several things:
-another ethanol precipitation and wash on your DNA
-during the pcr, first denature for 5 min (94 degrees) and then start the pcr
-you can make the annealing temperature 50 degrees or less for the first 5 cycles and then the other cycles as normal cycles
-add 1 ┬Ál of DMSO to your pcr mix.
and off course the things that were already said in the topic.
If you ever need an enzyme with proofreading (if you need to make overexpression constructs or knock-outs e.g.) I can recommend Phusion, worked really well on fungal genomic DNA.

-aspergillie-

Having DMSO while amplifying from genomic DNA helps PCR.

-scolix-

And I have to apologize, it's 1 microgram, not 1 milligram

-aspergillie-