I am trying to amplify a fragment of CMV genome from a dried blood spot (Guthrie card) but all I see in agarose electrophoresis gel are primer dimers, no expected band even in positive control.
It is a nested PCR technique, it has been used hundreds of times in our laboratory but now it seems it is not working anymore. The master mix was already made by a colleague who optimized the tecnique, it worked a couple of times but suddenly no amplification fragment is obtained.
I ordered new Taq polymerase, I made fresh new master mix, I repeated DNA extraction but nothing seems to work.
Did you order new primers or made a fresh working dilution from stock?
Hi,
Are your primers ok? By freeze/thawing the master mix you could be encouraging degradation of your primers. Ordering new primers may help. Also, after DNA extraction, are you nanodroping your DNA to ensure good quality? You may also need to play around with the annealing temperatures.
Good luck!
Thank you for your quick answers!
In fact, the only thing that is not new are the primers. We have two sets of them and both worked until now, but the truth is that the person who was in charge of this tecnhique didn´t aliquoted them. So the freeze/thawing-related degradation may be the issue that is blocking the PCR.
Since I have verified the thermal cycler program and other colleagues are using the machine with no problems reported, I say I have to blame it on the primers.
I will order a new set of them and then I will tell you how it went.
Thank you again for your help!
I would also recommend dissolving the primers in TE not water . Water absorbs co2 from air and becomes acidic and depurinates the primer. Aliquot the master stock so that you do not need to freeze thaw very often
what do your negative controls look like....if you have primer dimer contamination it will amplify really well and you will not get a proper product. Have you got a picture of the gel
Thank you Paul for your answer. Please find attached the picture of the gel. Can I dilute the primers in TBE? We don´t use TE in our lab, since we are a Clinical Pathology laboratory.
TBE is fine...anything that is alkaline buffered and has edta to remove magnesium which inactivates nucleases. Thank you for the gel picture. It is better to run gels for longer generally so that the size standard separates but there is quite a strong P-D band below the standard . P-D is very small so does not trap much ethidium bromide so when you see a band fluorescing it really does mean that there is a great deal of it compared with a pcr product band which are larger and, therefore, of stronger intensity. All of your tracks have this so either your primers are bad ( but this is not the case as they have worked well previously) or something has changed in your pcr like the annealing temperature has been lowered or the enzyme has changed from a hot start enzyme to a standard enzyme or the pcr mix is being left for a long time at a low temperature before cycling or the concentration of primers has increased dramatically but in a clinical lab I think that operating procedures have not changed . So I am still thinking that you have a p-D contamination problem in the pipettes, working area, water or some common factor. . If you have ordered new primers I would make them up in another lab or,at least, another workers area using their pipettes and plasticware. Meanwhile it would be interesting to set up a pcr in another lab or another working area again with their pipettes and ,ideally, their pcr mix to see if the p-d problem still remains. It would be useful to see if the problem is contamination or whether the current pcr conditions are causing the p-d
Check your master mix with another set of primers from a product you know it works. This in order to know if the others components in the reaction are OK (dntps, MgCl2, Taq pol). DNTPS are degraded easily due to temperature variations, whereas primers support more temperature changes.
Thank you Fernando. The dNTPs are the same we used to set the first master mix, probably I´ll order a new set of them too.
To remove primer-dimers, annealing temperature can be increased.
poor quality of template DNA or low primer concentration can also be a factor.
Please check your thermal cycler. I had similar experience before as all my pcr suddenly become not working at all, and it turn out to be the pcr machine heating blot temperature had run off.
The DNA template quality is not an issue, I performed a real-time PCR with the same template I used for the end-point reaction and the positive control and one of the samples were amplified.
The master mix is optimized, it has been working for more than 10 years, so I can say that the problem must be the reagents or the thermal cycler. Since there are other technicians using the PCR machine with no problems reported, the reagents seem to be the issue here. I am waiting for the new ones I ordered, I will let you know when I set a new reaction. Thank you for all your answers!
The new primers and dNTPs have just arrived. I have to dilute the primers but I have one more question: should I dilute the primers with TBE 10x or TBE 1x? By logic, since EDTA may inhibit Taq polimerase, it should be TBE 1x but since it is the first time I am doing this (and my colleague used to dilute primers with water) I want be sure.
If the new reagents don't work, you could have a problem with the water in the master mix. New ddH2O might work.
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