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Digest genomic (eukaryote) DNA before running PCR, is it necessary?


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6 replies to this topic

#1 phucvn

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Posted 14 February 2009 - 04:28 AM

Dear all,

I got problems with normal PCR amplifying genomic DNA. My genomic DNA is good in quality (Nanodrop check) but show no band on 1.5% gels. I tried PCRs with different primer sets designed carefully, especially ones for 18S rRNA (which is easy to amplify), but obtained no band or just smears.
My colleague suggested that because gDNA is too long that can not move in 1.5% gel electrophoresis and is difficult for the amplification.
When I broke gDNA with restriction ezymes and sonification, I got weak bands. I used Takare ExTaq PCR kits.
I am appreciated any of your suggestions.
Thank you

#2 perneseblue

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Posted 14 February 2009 - 06:55 AM

A nanodrop reading only tell how free your sample is from contaminants. It does not tell you the quality of your genomic DNA.

Thus a good nanodrop reading, could occur in a the genomic DNA sample that was free of RNA and protein contamination but completely degraded to 500bp lengths.

I would say try running your genomic DNA sample on a gel. If the sample is good, you should see only a single high molecular weight band >20kb.

1.5% agarose is too dense. Try a 1% or a 0.8% gel.

I would not shear/cut the genomic DNA sample. This can easily cut up your desired template sequence into un-usable fragments.

It is often the case that the gDNA sample after extensive purification still contains nonDNA contamination. These contaminant can inhibit the PCR. The trick to get around this problem is to dilute a sample of the gDNA with water and use the diluted gDNA for the PCR reaction. Try a dilution of 1:20 to 1:50. PCR is good enough to amplify samples this dilute.

Lastly, genomic DNA will melt fine. What are the cycling conditions are you using? How big is the desired PCR product.
May your PCR products be long, your protocols short and your boss on holiday

#3 hanming86

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Posted 15 February 2009 - 08:36 AM

U could try playing with dilution of the genomic DNA with the purpose of diluting out the PCR inhibitor.

Sometimes, doing an EtOH prep solves the problems at least in my experience. I dealed with PCR for 16S rRNA though not 18S.

Then you could also play around with the MgCl2 conc.

then you could also play with DMSO , Betaine and glycerol ...

Quite a lot of ways to work around this.
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#4 gebirgsziege

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Posted 15 February 2009 - 11:35 PM

Usually you should not have PCR problems with the 18S if your sample does not contain inhibitors or is strongly degraded.

But stringent PCR conditions are of high importance!!! Have you tested your primers before and know that they work or are they specifically designed for your organism ansd you should test them? Even the most sorrowly designed primers can fail sometimes! But maybe something is worng with them....we had a similar problem with spec. primers where the company messed something up.....we got a new batch and everything started to work.

I have one more suggestion: try a nested PCR with universal primers working for your region and try a nested PCR with your sample.

But maybe knowing more about your organism and what you want to do with the PCR products can help us to give you a more specific tip :blink:
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#5 molgen

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Posted 16 February 2009 - 02:42 AM

You do realize that 18S is RNA and not DNA.
There for you need to first do reverse transcription with random Hexamer (not OligodT!!!) to get cDNA. Only then can you do PCR for the 18S rRNA.

#6 WHR

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Posted 16 February 2009 - 06:36 AM

You do realize that 18S is RNA and not DNA.
There for you need to first do reverse transcription with random Hexamer (not OligodT!!!) to get cDNA. Only then can you do PCR for the 18S rRNA.


I think he meant the gene for the 18S rRNA, not the RNA itself.

Edited by WHR, 16 February 2009 - 06:37 AM.


#7 phucvn

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Posted 21 February 2009 - 10:21 PM

Dear all,

Thank you very much for your suggestions. I finally solved the problem. I purified genomic DNA, made new working solutions for primers. It is quite difficult for me to fix the program for several degenerate primers in a multiplex PCR but it works. I need DNA fragments to clone and sequence them to compare with cDNAs. That is my purpose.
I hope to return here again with different topics.
Cheerfully,




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