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Real-time PCR does not work, while the conventional PCR (with same conditions an


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#1 Procyon

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Posted 12 February 2014 - 01:01 PM

Hi, The problem that i'm having is that the qPCR is not amplifying (I made an agarose gel electrophoresis with the qPCR "products" to check for amplicons, but nothing), yet I have tested the program, same components concentrations, same DNA aliquots, in a conventional PCR, and it works. The only difference is the addition of SYBR green I listed here.

 

 

I used 0.125 ul from a 100x aliquote. Is possible that this ammount of SYBR green inhibits the amplifycation?

 

SYBR® Green I Nucleic Acid Gel Stain - 10,000X concentrate in DMSO: https://www.lifetech...ID=search-s7563

 

 

Attached File  aprA qPCR primero 7 febrero.PDF   220.4KB   61 downloads


Edited by Procyon, 12 February 2014 - 01:14 PM.


#2 Curlis

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Posted 12 February 2014 - 03:04 PM

Hi, 

 

Even I had the same problem as you are facing right now. Did you run your sample in gel after qRT-PCR. You should be able to see band as it was in RT PCR without quantitative (basically normal PCR as you do but you use cDNA here). If you doesn't see a proper band or nothing at all then might be problem in your machine. 

 

if you see a product that means everything is fine, but the problem in SYBR GREEN dye. You need to change it. Even when I changed it then everything was working great. I hope it should work. 

 

PS: Whenever you dilute SYBR GREEN in TE' buffer then keep it for not more that 2-3 weeks and than make a fresh vial of dilution.  



#3 Procyon

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Posted 13 February 2014 - 10:03 AM

Hi, 

 

Even I had the same problem as you are facing right now. Did you run your sample in gel after qRT-PCR. You should be able to see band as it was in RT PCR without quantitative (basically normal PCR as you do but you use cDNA here). If you doesn't see a proper band or nothing at all then might be problem in your machine. 

 

if you see a product that means everything is fine, but the problem in SYBR GREEN dye. You need to change it. Even when I changed it then everything was working great. I hope it should work. 

 

PS: Whenever you dilute SYBR GREEN in TE' buffer then keep it for not more that 2-3 weeks and than make a fresh vial of dilution.  

 

No, no reverse transcriptase (RT) reactions involved. I hoped that was a problem of SYBR Green, but no. The DNA used as template was extracted from an environmental sample. As I told before, the conventional PCR works, giving me the unique products of the expected size; but when I run a gel after the qPCR I got nothing; no bands, no fluorescence.

 

The situation is that everyone in the building (not just my lab) use that machine, and it works. Someone told me the check the SYBR Green datasheet looking for any indication of the maximum amounts that do not inhibit the  amplification (PCR), but I heve not found anything. Other fact is that I'm not using a qPCR kit (proved to work for other people here), but the same Taq-polymerase, MgCl2 and it's buffer I use in conventional PCR.

 

 

I'm giving details until someone finds something weird.

  • Reactive                   [stock]                [use in Rx mix]                           Volume per reaction
  • H2O                                                                                                         14,5  ul
  • Buffer                         10x                      1x                                               2,5  ul
  • MgCl2                        50 mM               1,5 mM                                           0,75 ul
  • dNTPs                       10 mM                0,2 mM                                          0,5 ul
  • Primer Fw                   10 uM                0,3 uM                                          0,75 ul
  • Primer Rv                   10 uM                0,3 uM                                          0,75 ul
  • Taq Pol                       5 U/ul                0,025 U/ul                                    0,125 ul
  • SYBR Green              100x                                                                      0,125 ul
  •   DNA                         2,3 ng/ul            11,5 ng                                           5 ul

                                                                                                                      Total volume of  25 ul per reaction

 

 

Take notice that the SYBR Green is SYBR® Green I Nucleic Acid Gel Stain - 10,000X concentrate in DMSO, and I'm using at a final concentration of 0,5X. I have been told that these concentrations of SYBR Green for a Gel Stain are not the same for qPCR; so is it possible that there is an excess of DMSO that inhibits the qPCR???


Edited by Procyon, 13 February 2014 - 01:09 PM.


#4 Procyon

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Posted 19 February 2014 - 07:56 AM

I made a conventional PCR with all the real-time-PCR condictions and with added SYBR Green.

 

The results confirmed that there is something n the SYBR Green that inhibits the amplification of my gene of interest. I don't know if I should try the qPCR with less SYBR green or using a kit.






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