We carried out nested PCR for bacterial diversity studies in coconut rhizosphere soils. Total DNA from soils were extracted and an external PCR was done using primers set f27/r1492 for the full length bacterial 16S rDNA. Then we used 338f-GC/518r primers for internal PCR to amplify v3 region of bacterial 16S rDNA and got two closely located bands appearing at the expected position (220bp) from the soil DNA. We repeated the second PCR with different conditions but always got two bands. A positive control using DNA extracted from bacteria however gives a clean single band (gel picture attached)
Would anybody help us to know why we get two bands? Is the second one (below) from archae? Can we use the amplicon for DGGE run? Or we must alter PCR condition until we get a single clear band and then run the DGGE.
Thank you.
I am sure you double check this but is your negative control negative? I can’t see the negative control on your gel. And did you keep using the negative control from the first PCR for the second PCR (in addition to the negative control you would normally do for PCR) to make sure you do not have any contamination?
Did you try to do your second PCR directly on the extracted DNA? Why do you do a nested PCR? I would expect the primer pair 338-GC / 518R to work fine directly on the DNA.
You can’t do your DGGE if you do not have a single band; you need to solve the problem first. My advice would be not to do a nested PCR which can be problematic and increase the chance of having multi bands... So try to do your 338-GC / 518R PCR directly on the extracted DNA.
As Aimeric Blaud says, I'll try with the DGGE primers directly on the DNA, I always do that, for both bacteria and fungi.
However, I have used another cycling conditions:
Denaturation : 94ºc – 5min
: 94ºc – 30s
Annealing : 57ºc – 45s
Extension : 72ºc – 45s (35 cycle)
Final extension : 72ºc – 10min
Ref.:Grossman et al 2010 _ Amazonian Anthrosols Support Similar Microbial Communities that Differ Distinctly from Those Extant in Adjacent
Thank you Aimeric, Dulce and Biswarup.
To Aimeric,
Ö yes, your are right, our gel does not have negative control.
Ö yes, we have carried out PCR using DGGE primers directly on soil DNA and still got two bands (gel picture attached).
To Dulce,
Ö we will try the PCR conditions you mention and let you know the results.
To Biswarup'
Ö have emailed to you
Aimeric,
the second ppt slide shows the result of direct PCR with DGGE primers on soil DNA.
Thanks
Doing the PCR directly with 338-GC / 518R primers of the DNA improved a bit the results (no bands on the top part of your gel in comparison to the nested PCR one) even if you still having 2 bands around the desire product size.
I would perform a gradient PCR to determine the best annealing temperature or try the protocol suggested by Dulce Flores-Rentería. You can have these 2 close bands if the annealing temperature is not correct (I already witness this problem because of the annealing temp).
Let us know if you manage to solve the problem
Best,
Aimeric
You may also want to consider:
> Longer final elongation time (5-30 minutes of 72ºC)
> Slow touchdown to 4C after final elongation stage
> Check primer degeneracy
> Concentration of DNA added to reaction
> number of PCR cycles.
· Janse I, Bok J, Zwart G .2004. A simple remedy against artifactual double bands in denaturing gradient gel electrophoresis. Journal of microbiological methods. 57:279-281.
Hi dear Murali Gopal
I have exactly the same problem in my research
How was the problem solved?
Please help me
thanks
And about the quantities of the PCR components for 25uL?
I use these primers for traditional PCR (not DGGE) and I guess that some quantities is wrong.
I use: 16.4 uL of H2O for PCR
2.5 uL of Buffer 10X
1 uL of dNTP Mix
0.5 uL of each primer
2 uL MgCl2
0.1 uL of taq
2 uL of DNA
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