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PCR - template dilution

PCR - template dilution - PCR - Polymerase Chain Reaction Forum

PCR - template dilution - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 01-14-2011, 09:59 AM
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Default PCR - template dilution



Hi all,

I have to do PCR to get product from one allele (to get homozygocity from heterozygous insertion). In an original article (Vogelstein & Kinzler 1999) is ment that the amount of that template is approx 1,5 pg DNA. But in my condition I have problem visualise product (ethidium bromide) when I use 25 pg of template (still heterozygous).

Please, can somebody advise me how I should change the conditions to successfully finish PCR? Thanks a lot!

My conditions:

for 25 ul volume:
10X Taq Buffer (with (NH4)2SO4 and MgCl2)..... 2,5 ul .... 1x conc.
dNTPs (2mM mix) ........................................ 2,5 ul ..... 0,2 mM
primer set (10mM each) ............................... 1,0 ul ..... 0,4 pM
Taq DNA polymerase (5u/ul) .......................... 0,1 ul ..... 20 mU
template .................................................. . 1,0 ul

cycler program: 94°C 2 min; 38 cycles: 94°C 20s, 60°C 30s, 71°C 40 s; 71°C 3 min.
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Old 01-14-2011, 07:19 PM
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Default Re: PCR - template dilution

In your primer set final conc. you mean mM, right. And your final Taq conc. should be 0,5 U ( 1 ul of Taq = 5 U, then: 0,1 ul of Taq = 0,5 U, regardless of final volume).

Since template conc. seems to be your only problem you could rise the concentration of template by rising the volume you add. Try doing a gradient of template conc. or volume to standarize the reaction. For example, if 1 ul isn't working try adding 1.5, 2, 2.5, etc. Also, when doing this try to use the same DNA and the same conditions, working the different volume tubes in duplicates should help too.

Hope it helps
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Old 02-04-2013, 09:13 AM
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Default Re: PCR - template dilution

PCR is very versatile. Many types of samples can be analyzed for nucleic acids. Most PCR uses DNA as a target, rather than RNA, because of the stability of the DNA molecule and the ease with which DNA can be isolated. By following a few basic rules, problems can be avoided in the preparation of DNA for the PCR.
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