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PCR-One sample works the other does not?


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#1 Myriam

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Posted 06 November 2004 - 12:53 PM

Hey, I am new, but I hope somebody can help...

I am trying to run a PCR on gill samples since 6 months. I finally got it to wotk after changing annealing temperature, Taq and dNTPs.
Now I have 9 god-looking genomic DNA samples, which all have around the same concentrations and look nice on the gel, but only one works!!!!!!!
What am I doing wrong? I retried using the same samples again with different amounts of DNA, but nothing works! It just does not seem normal!

If anybody could help, that would be awsome!!!!! :(

Thanks

Myriam

Edited by bioforum, 07 November 2004 - 03:14 PM.


#2 madhubiotech

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Posted 06 November 2004 - 09:22 PM

hi

ur PCR seems alright. even i faced the same problem sometime back with reference to someother samples of plant DNA. what you can do is try changing the concentration of Magnesium in the buffer. u can prepare a cocktail of different concentrations of the buffer and then try the reaction. may be it will work. i did like that and it worked for me.
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madhan shankar
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Dept of Biotechnology
DR NGP College
Coimbator35
India

#3 bob1

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Posted 07 November 2004 - 12:55 PM

Hi

What are your 260/280 absorbance ratios like from when you quantitated the DNA, if they are close to 1.8 then your DNA is pure, but if lower than about 1.7 or higher than about 2.0, then you have contaminants in the extraction, these could be inhibiting your PCR. Solution: try re-extracting the DNA on fresh samples or try cleaning up your already extracted DNA, there are a range of kits to do this.

Hope that helps

#4 Myriam

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Posted 08 November 2004 - 10:41 AM

Hi veteran

The ratio is fine, it si aroun 1.7 to 1.8. Not the best, but it should work, right?

I will try changing the Mg conc., and will see what happens.

Thanks

Myriam




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