Few weeks ago I was having troubles with my PCRs, sometimes they work, sometimes don't, so I performed a control PCR in order to check if everything was ok with my conditions and reagents. As a result, everything works and I get my bands as expected.
However, now I'm trying again the same conditions, the same reagents, the same quantities, the same control DNA template, the same thermal cycler (which is working for other people), also I'm vortexing every time and working on ice, and nothing, no bands anymore. I can only see a perfect ladder on the gel.
I don't know what else I can do except for staring my notebook. I'll be thankful for any suggestion.
From what you've explained, there seems to be some contamination in the PCR components. Another round of debugging seems like a good idea.
Do the easy/cheap stuff first - change the water, prepare new working solutions of the primers, check the DNA on a gel.
If things are fine there, start swapping out all other components.
Also if you have any doubts that the plasticware you're using might be contaminated, it would be best to get new ones from a fresh package.
Situations like this are really frustrating, so I hope you find the solution quickly.
Hi Sara Mena-Bueno
I think which you could try a quantification of your primers (to accurate the concentration) and your DNA (to assess the quality and quantity). In another hand, are these primers recently designed? Probably, also might have a bioinformatic issue on the primer design. Think it.
More informations can lights on your way.
Best wishes,
Could it be the samples...?
Could you add more template DNA to the Rxn?
They don't have Proteinase K, in them do they?
I have seen this with my Genotyping gels if the PK is not fully denatured after digesting the tail biopsy, is so re heat before PCR.
Add more DNA sample to your PCR Rxn..?
Best Wishes!
That's difficult. And it stopped working with the control DNA sample?
What are you samples exactly? cDNA or DNA? And what is the source?
We'll need more details about your protocol and your samples to start troubleshooting.
The last time this happened to a friend of mine, it was a combination of issues. I had to redesign her entire protocol, including the cycle temp and time, and change all her reagents.
Hi,
Sara,
I would suggest a few things:
1. Try to extract DNA afresh from fresh samples (Multiple freeze thaws degrade DNA).
2. Your material is diffrent from other person's material, so it will show slight variation. If your material (from which you are extracting DNA is rich in phenolic compounds and other inhibitors), then try diluting your template like 1:10 or 1:100. Decide dilution only after checking the concentration of DNA.
3. Try using BSA in PCR rxn.
Hope this works!!!!!!
From what you've explained, there seems to be some contamination in the PCR components. Another round of debugging seems like a good idea.
Do the easy/cheap stuff first - change the water, prepare new working solutions of the primers, check the DNA on a gel.
If things are fine there, start swapping out all other components.
Also if you have any doubts that the plasticware you're using might be contaminated, it would be best to get new ones from a fresh package.
Situations like this are really frustrating, so I hope you find the solution quickly.
Hi, this may be as a result of contamination as PCR is highly sensitive technique.
Check primers concentrations and have a look to Mg++ concentrations. Try with a dilution of your DNA. High quantities of DNA can inhibit PCR reaction
Hi Sara,
How did you perform the extraction of nucleic acids for your PCR?
A common problem is that for example in the classical extraction (no kits used) some reagents (ethanol, etc) can interferer with downstream applications due to the fact that the nucleic acids are not properly clean and residues of those reagents are still present
Good Luck!
hii ....sara
i would like to give some suggestions
1)First check the quality of your control DNA samples by Nanodrop and 0.8% of agarose gel run
2) vortexing every time for reagents and primers were fine but don't vortex the DNA Samples. it interfere amplification of PCR reaction .
3) check the method of extraction if manual method maximum time ethanol can interfere the downstream process.
Hi Sara,
I suggest to check the design of your primers, they might be making self or hetero-dimers. And also you should check them by gel electrophoresis your aliquots might be degrading.
Good luck¡
Hello Sara,
After going through the problem you are facing, i would recommend you to use the PCR product as template for a second round of PCR ( basically Re-PCR) or increase the cycle number, as there might be really little amount of DNA present in the PCR product.
If you find out primer dimers in the bottom of lanes, i fear the primers are not working, and you need to do something to fix them.
I think this might solve your problem.
Good luck.
Thank you all very much for your answers! I've finally achieved to have PCR product, just changing to new buffer, MgCL2 and polymerase stock, so maybe some of them were degradated, or it has been result of chance. If I start facing problems once again I'll try some of the many solutions you've gave to me.
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