PCR product help please

[Rabar Mantil]

Hi

I am a PhD student and stuck with the PCR no band in gel. so basically I am looking to have a band size 1250 bp

Using Master Mix

1 microliter Primer R 20mM
1 microliter Primer F 20mM
5 microliter Buffer 10x ( MgCl2 added by 1:1)
1 microleter dNTP 10 mM
1 microleter DNA template ( 448 ng)
1.5 microliter Taq polymerase
29.5 microleter dH2O

95 for 4 minute
95 for 45 sec
55-60 for 1.20 minute
74 for 1 minute
gradient 35 cycle

74 for 4 minute


The gel is 1 % TBE

110 Volt , 200mPA and 30 - 45 minute

My results are no band as in the picture

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[Microbug]

Does your primers actually work... as in do you have a positive control for the primers? Or if you have controls for the PCR reactions itself?

 

[Rabar Mantil]

Does your primers actually work... as in do you have a positive control for the primers? Or if you have controls for the PCR reactions itself?

Two DNA templates supposed to be a positive control for the primers, but still no luck with them as well.
The primer are degenerated , that has been designed based on both of them

 

[TurtleGnome]

The primer are degenerated

^not sure if you mean "generated"? But it sounds like a problem with your primers.

 

[Microbug]

Two DNA templates supposed to be a positive control for the primers, but still no luck with them as well.
The primer are degenerated , that has been designed based on both of them

Hmm... I would perform a PCR reaction with different primers that has worked in the past to control for the PCR reaction itself. If that works, then you know its not an issue with the way you set up your PCR and it is most likely the primers.
Is it your first time using these primers, did they work in the past?

 

[Microbug]

The primer are degenerated

^not sure if you mean "generated"? But it sounds like a problem with your primers.

degenerate primers is a thing.

 

[TurtleGnome]

Ah, yes, the phrasing confused me. My mistake

 

[Rabar Mantil]

Its my first time using these primers as I had to design them , because its not been used previously . My thought is why it reacts differently with all three DNA template,, each 12 wells are for one organism,, so the primer looks like binding in the last 12 well bottom,, but did not produce a real band.

 

[Microbug]

Its my first time using these primers as I had to design them , because its not been used previously . My thought is why it reacts differently with all three DNA template,, each 12 wells are for one organism,, so the primer looks like binding in the last 12 well bottom,, but did not produce a real band.

To be honest, it doesn't look to me that it's binding even though it looks different...

 

[tr]

Its my first time using these primers as I had to design them , because its not been used previously . My thought is why it reacts differently with all three DNA template,, each 12 wells are for one organism,, so the primer looks like binding in the last 12 well bottom,, but did not produce a real band.

How did you design the primers? Did you use an appropriate program and run an in silico PCR before you ordered the primers?

If your positive control is not working that suggests either your primers are not correctly designed or the conditions for your reaction are wrong.

What's weird is that you are using a pretty low annealing temperature and a gradient PCR protocol, which by the later cycles should get really nonspecific binding and start to give you some random smeary bands at the bottom. But you have nothing there. Your gel is super clean. Are you, uh, sure there is template in there?

Is your gradient running from 60 to 55? If so what are the suggested ideal annealing temperatures for each of your primers? You could try running a bigger gradient, getting to very low annealing temperatures by the end. You should get a lot of junk amplification but at some point you should see your band of interest as well.

If the in silico PCR works the next step is to troubleshoot by setting up a range of different PCR conditions with the positive control vector as template. Vary the amount of template and the annealing temperature range, and it would be fair to include a positive control with a different set of primers that are known to work for a different target sequence in the same vector. Some people try adding additives like DMSO but in my experience that's usually pointless. The PCR works when you have well designed primers, clean template, and the correct annealing temperature.

By the way this isn't really the right forum for this question. Try a forum specifically for bioscience troubleshooting, like biotechniques, biocompare, scientistsolutions, or protocolonline.