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I am a PhD student and stuck with the no band in the gel results.

I am using the following

  • 1 microliter Primer R (20mM)
  • 1 microliter Primer f (20mM)
  • 1 microliter dNTP (10mM)
  • 1 microliter DNA Template ( 448 ng)
  • 1 microliter TAQ polymerase
  • 5 microliter Buffer (10x) added MgCl2 by 1:1
  • 30 microliter PCR grade water

Program:

  • 95 for 4 minute
  • 95 for 45 sec
  • 55-65 for 1.20 minute
  • 72 for 1 minute
  • Gradient 35 cycle
  • 72 for 4 minutes

Gel:

110 volt, 200 mPA for 30 - 45 minute

I get PCR products as in the images. There are no bands while there are intense bands at the bottom and some of the wells.

My question is, is that at the bottom products?? what can be done to make them real? If not then why there are differences between the two lane of wells. Each 12 well are representing a one type of DNA template (I used three different organisms DNA).

enter image description here

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    $\begingroup$ First: Your PCR is working in principle, the bands at the bottom are beautiful primer dimer bands which indicate this. Then: What is the source of your DNA and has it ever been user successfully for PCR? How is it prepared? $\endgroup$ – Chris Aug 9 '17 at 18:48
  • $\begingroup$ Hi Chris, thanks for your reply. The DNA are from LLactobacillus and Lactococcus bacteria. The primer has been designed as degenerated based on both of them. Up to now I couldnt get the PCR work . $\endgroup$ – Rabar Mantik Aug 9 '17 at 18:56
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    $\begingroup$ Ladder looks good... $\endgroup$ – Willk Aug 9 '17 at 23:34
  • $\begingroup$ Can you post the sequence you intend to amplify and the sequences of the primers you are using? Also, do you have any positive control for the Taq? $\endgroup$ – alec_djinn Aug 10 '17 at 8:19
  • $\begingroup$ Do you use genomic DNA? Then you can try to enhance the outcome by using either less template or add additional MgCl2. You can also try different PCR methods (i.e. spiked PCR or Touchdown). $\endgroup$ – SeRe Aug 10 '17 at 12:11
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Use control primers targeting something which for sure gives a product. If you have none, ask your colleagues or take some out of literature. Make sure your product is small enough to be amplified in the time given and check for the correct band with a restriction enzyme.

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I'm not sure about the scale of your ladder but regardless the DNA causing the banding is very small. They are most likely primer dimers. I would check the primer sequences to make sure there isn't a lot of complementarity. You can also try manipulating reaction conditions like concentration of magnesium chloride or the primers themselves etc. I would run an optimization set of PCRs with variable reaction conditions.

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My first guess would be that you're using too much template DNA for these conditions. Your wells light up quite a bit, 448 ng is also on the high side.

Try lower concentrations of template DNA (1-100 ng), perhaps only with a 55 °C annealing step. At the moment you don't have any product at all, so I wouldn't worry about non-specificity yet (and 10 °C is also quite a small range to fix that).

Other things that stand out:

  • I assume it's 20 uM primer, not mM
  • You're adding too much buffer
  • It looks like you're making each reaction individually (looking at the template amounts in the first 12), I suggest making a master mix and aliquot that out.
  • You have quite some space to run your gel longer, that might be very useful if you do get some product.

You should have a bit more information as well. What's the size of the products that you expect? Do you use high GC organisms? Did these primers work before for other people? Did the genomic DNA work before for other people?

NEB has a lot of useful information about PCR and other DNA work on their website, check it out: https://www.neb.com/tools-and-resources/usage-guidelines/guidelines-for-pcr-optimization-with-taq-dna-polymerase

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What you can try is different buffer (10 x) solutions, also with or without MgCl2. In the lab where I used to work with PCR, we had a panel of different PCR buffers in the freezer. We tested some of them, to see which one fitted best (hence which one gave the right size band). Just like your temperature gradient, to see which one performs best.

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You can try a touchdown PCR. It is often helpful when no band is present.

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