What is the problem with the analysis in the LightCycler 480? Two-step cycling?

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No amplification data for qPCR on MxPro?

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25 February 2021 8,039 1 View

Problem with ligating 2.8 kb insert into 8.5 kb plasmid?

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Color of spiro-ometad film?

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21 February 2021 1,831 4 View

Has anyone encountered any problems with abm good microRNA primers for quantification of mature miRNAs?

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QPCR tubes for Roche Lightcycler Nano?

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Does the property of GFP affect a fusion protein's localization?

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How do you prepare a standard dilution for qPCR?

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Barry Atkinson

Hi Patrycja,

I haven't used the LC480 in a while (and even then I used hydrolysis probes rather than SYBR) but I think the answer lies in the fact that you haven't told the machine to look for fluorescence at any point. I would imagine that you have to set the acquisition mode to something like 'single' after one of the amplification steps - if you do so a little green dot will appear on the temperature plot telling you when a reading will be taken (you'll see 45 appear at the marked point in each cycle). From the one or two times I did run SYBR assays, I think you also have to set to 'continuous' on the melt curve part of the programme in order to see what is happening here

Hope that helps

All the best

Barry

Patrycja Ogonowska

Thank you very much for answer! You were right. The problem was in preparing the conditions in LightCycler 480 Software. In the last step of amplification in "Acquisition mode" window I chose "none" instead of "single", so the program couldn't read the fluorescence after the each cycle. Now, everything works properly. Thank you for your help!

No worries

Sreekala V Sathyavruthan

Patrycja Ogonowska ,

Hello Patricia, I had the same problem today, couldn’t analyse the result On lightcycler 2.0.

So, how did you solve this? Did you do exactly what Barry Atkinson advised you to do? Please advise.

Many thanks.

Hi! Yes, I conducted my qPCR experiments according to Barry Atkinson advise and it worked. You have to rember that in amplification program, the last step should have the status "single" in the acquisition mode. Then the fluorescence will be read after each cycle. I recommend three step amplification than two step, to avoid primer-dimer formation. I attached a screen of full run program dedicated to SYBR Green dyes.

Good luck!

Thanks very much patrycja. I really appreciate your help.I will try this.

Its a commercial kit that I am trying and they advise two step rather than three so I might have to stick with it. I will be developing more assays so I will keep your advise regarding this.

Sree.

Carlene Chinn

Patrycja Ogonowska Hello, I know this was posted some time ago. I believe I made the same mistake. I modified the amplifications but left it as None and not Single. Did you have to rerun the entire plate or can this be changed still for analysis?