I conduct qPCR to determine the number of genome-containing particles in an AAV preparation using SYBR green technology. We used AAV-GFP plasmid as standard, and make 5 serial dilution (1:5) and triplicate, started from 8.7E10 copies and so on. However, I didn’t get a decent amplification curve and its appear as a junk.
I used to do the same experiment 2 months before and I never face such problem. I try new reagents like SYBER green, DNase, enzyme buffer and primers, I was thinking maybe problem with reagents. But still face the same problem. Furthermore, I checked the concentration of my standard (AAV-GFP) by Nanodrop and it was okay. as for the serial dilution are concern, I make them very carefully, vortex and pipet up and down several times. I think my serial dilutions will be okay. To sum up, I didn’t find the exact cause of this junk amplification curves.
Here I attached the amplification curve, melt curve and raw data plot of my standard curve.
Any idea what might have caused this?
Thanks,
Regards.
Hi Waqas Nawaz , Welcome to visit our website to get more information: www.genemedi.net.
Your result is obviously a failed QPCR experiment. There may be various reasons, but it must be that one factor has completely failed.Two things should be confirmed first，fist one is to QPRC for another common gene such as Actin or GAPDH that you usually used, to check whether your whole QPCR system was OK, including the reaction mix, the machine, etc,.；the other one is to check that your AAV plasmid, better to re-preparation of the plasmids, and use a new synthesised ITR primer with Taq polymerase to do a normal PCR, and make a gel electrophoresis with this PCR product to check the band size and concentration.
Hope this would help you out
New reagents like DNAse?
What is the purpose of DNAse, here? Sorry if I'm being stupid, but I would think a DNAse is one of the few things you absolutely don't want to have hanging around when you're dealing with DNA viruses, plasmids, and PCR.
John Hildyard DNase we used for our sample virus not for the plasmid. To measure the titer of assembled virus particles in our harvested production cells, we first digested all cellular and DNA left in our virus stocks. We therefore treated 5 µl supernatant from pelleted cell lysate with 10 µl DNase1 and 5ul enzyme buffer and 30ul DPEC water, incubate at 37°C for 30 min, 65C for 5mint to inactivate enzyme.
Waqas Nawaz , Just as a question, do you think there is a potential for DNA contamination in your stock? It seams that according to the melting curve, You have at least two amplified products.
I have also a question related to my work! If I understand correctly, your samples are gDNAs, and you use the plasmid serial dilution as standard. Is your method absolute quantification? If so, please can you tell me if the standard curve fitted with the results of the gDNAs in your previous work and got the correct results?
Hi Waqas Nawaz , Welcome to visit our website to get more information: www.genemedi.net.
Your result is obviously a failed QPCR experiment. There may be various reasons, but it must be that one factor has completely failed.Two things should be confirmed first，fist one is to QPRC for another common gene such as Actin or GAPDH that you usually used, to check whether your whole QPCR system was OK, including the reaction mix, the machine, etc,.；the other one is to check that your AAV plasmid, better to re-preparation of the plasmids, and use a new synthesised ITR primer with Taq polymerase to do a normal PCR, and make a gel electrophoresis with this PCR product to check the band size and concentration.
Hope this would help you out
Yes, it's a failed qPCR. I think melting curve information is wothrless if you have not amplification, just noise. Maybe you have to explain step by step your procedure, albeit if you conducted successfully in the past it should not be an error in the procedure (bad covering of the plate?). If you are not sure on your reagents and think could be your samples try to use stored samples which you know they worked in the past.
Roghayeh Shirvani no i dont have gDNA, i used dsDNA. Yes i serially diluted this dsDNA plasmid for my standard curve. i used absolute quantification.
in my previous results it was okay and i get a decent standard curve. Even my other collegue also practice the same method for AAV quantification and his result are pretty good.But i didn't sort out that whats may the reasons for my recent results.
Yixiong Chen thanks, sure i will visit it.
I used some AAV which i successfully quantified before, as a positive control. However, they also didn't give me an amplification curve, and appear as junk. The machine is also fine, because it a common and used daily, but no one complain. Furthermore, i also use other lab qPCR for confirmation, so i think the machine have no problem.
As for the plasmid is concern, i check its concentration by nanodrop. and further then i used this standard plasmid (AAV-GFP) for transfection to check it efficiency and i found by florescence microscope that its successfully transfect the cell line.
- Badges
- Science method