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Genomic DNA and PCR


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4 replies to this topic

#1 shimshady

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Posted 18 May 2009 - 08:20 AM

Are there any tips and tricks in doing PCR using genomic DNA? More DNA? Different PCR protocol? I tried once, maybe my primers are not any good and i can try and fix that but in the mean time i want to try other, different steps in the termocycler? Thanks

#2 klchisho

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Posted 18 May 2009 - 09:53 AM

Hi,
From my experience, I've found that up to 10ng of DNA works best, but I guess that may depend on other variables. This PCR recipe has worked well for me (a 20uL reaction):
Combine the following and mix well (vortex and spin down all reagents prior to use):
10X Reaction Buffer: 2uL
25mM MgCl2: 1.2uL
10uM Primers: 0.4uL each
10mM dNTPs: 0.4uL
5 U/uL: 0.2 uL
ddH2O: 8.4 uL (volume of water can be varied, depending on your DNA concentration)

Add 13 uL of master mix/sample.
Add approximately 7uL of 1ng/uL genomic DNA, spin down PCR tubes.

Make sure that you are using the correct annealing temperature for your primers. If PCR product bands are weak, you can try to increase the number of cycles, to start. Or, if you don't see any primer bands at the bottom of your gel, that may indicate that your primer concentration is too weak in your reaction.

Hope this gives you something to start off with! :)

#3 hanming86

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Posted 25 May 2009 - 10:51 PM

Dilution of genomic DNA sometimes work wonder. the dilution gets rid of the inhibitor present in the extracted DNA which then improve the success of PCR.

for Genomic DNA , it's advisable to have denaturation time of at least 1min.

10ng/ul of gDNA is sometimes suffice for a PCR reaction.

i usually use mastermix to prevent pipetting error and all that. save time and energy anyway. not an issue of laziness, just an issue of consistency that i prioritize
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#4 warsel

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Posted 06 June 2009 - 02:30 AM

The main thing that has already been mentioned is to have clean genomic DNA to work with (otherwise you will need to dilute).

Initial denaturation of 4min or so is also a good idea. I wouldn't do all the denaturations in the PCR for that long if it doesn't have to be, but going 10 cycles or so with 1-3min and then go down to shorter times should for the rest of the program work fine.

(after all you don't really copy off the genomic DNA after 4+ cycles any more anyways)

#5 shimshady

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Posted 10 June 2009 - 04:53 AM

The main thing that has already been mentioned is to have clean genomic DNA to work with (otherwise you will need to dilute).

Initial denaturation of 4min or so is also a good idea. I wouldn't do all the denaturations in the PCR for that long if it doesn't have to be, but going 10 cycles or so with 1-3min and then go down to shorter times should for the rest of the program work fine.

(after all you don't really copy off the genomic DNA after 4+ cycles any more anyways)



Is this because the genomic DNA is so big, it take longer denaturation to open up? I didnt think of that, it makes sense.




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