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Gel doubling banding and resolution problem


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#1 danibak

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Posted 27 February 2015 - 01:17 PM

Hi all,

I am having some issues getting good resolution bands from my PCR.  Originally my mistake was that my dNTP's were not dilute enough but I made new aliquots and started getting banding.  My bands are not that bright though and I have tried many concentrations of DNA (Full strength; 1:5; 1:10; 1:30).  I also tried messing with the dNTP dilutions (1:5; 1:6; 1:7; 1:8).  I also am getting double banding which for what I've heard means that there is to much dNTP but the catch is that on my last few gels its been on the more diluted samples.  I will post the picture of my most recent and probably my best gel.  Any suggestions would be taken kindly!  Thanks!  



#2 danibak

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Posted 27 February 2015 - 01:24 PM

I also forgot to mention that I am running a 1% Agarose gel in SBE buffer at 160V for 10 minutes.  I just need to get crisp bands, because we use this as a test of our DNA before we run it through DGGE.



#3 bob1

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Posted 01 March 2015 - 11:44 AM

How much of each component are you actually adding (useful if you can convert to standard units)? Dilutions might help, but it depends on how much is going into the reaction to start with.



#4 danibak

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Posted 03 March 2015 - 07:28 PM

Hi,

I am using 16.7 uL H20, 3.3uL Buffer(w/MgCl2), 2.5 uL DNTP(diluted, as mntioned above), .25 uL for primer, .75 uL rev primer, and .13 uL Taq per sample

 

I am following a protocol that people in my lab have used for years however I was just kind of thrown into and so I have the trouble of (re)trouble shooting everything.  Total of 24 uL per tube plus 1 uL of DNA (diluted, as mentioned above)



#5 bob1

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Posted 04 March 2015 - 01:20 PM

I was meaning how much DNA in terms of ng, how much dNTPs and primers in terms on moles (with appropriate prefix u, p, m, etc.)



#6 danibak

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Posted 05 March 2015 - 06:09 AM

Around 20 ng/uL of DNA.  Both the for and rev primer are 10uM and the dNTP are 2mM each



#7 bob1

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Posted 06 March 2015 - 01:34 AM

And the template type is genomic DNA or plasmid or...?

 

Often there are inhibitors in template that can be diluted out, but you usually need to do much greater dilutions than you are currently using (1:100 -1:10000), but typically starting DNA concentrations are much higher.






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