Hello
I used high fidelity enzyme in my PCR and I saw smear on agarose gel. I repeated the PCR with different amounts of cDNA. But I did`nt get a sharp band. what should i do to resolve my problem?
Is this a pcr that you have done before?
Are you running a no template control sample (water instead of dna) in your pcr
What are the annealing temperatures of your primers and what is the annealing temperature of the pcr cycle. How many cycles of pcr are you running
Zahra Sadat Eslami I bet you are doing RT-PCR. Your synthesis of cDNA might not be successful. That goes to that your RNA quality is not good enough? Did you check your isolated RNA on a gel for its integrity? (please show us the gel, if you do). Have you tried another cDNA synthesis from RNA (repeat again)? It is always good to attach your gel here for problem diagnosis. A picture worth a thousand words....
Dear Zahra Sadat Eslami
During setting up the reaction , please try to have a positive control, negative control and a no template control, that would confirm you that your pcr ingredients are working properly and if there is any problem with the cDNA that you are using. If evrything remains ok in the control sets then u play with the annealing temp. And conc. of template. But i would recommend you to prepare the cDNA freshly.
And while running the agarose gel, ensure that there is no DNAse comtamination.
check RNA quality on a gel
Check the concentration of the starting template. Make serial dilutions of template nucleic acid from stock solutions. Perform PCR using these serial dilutions
carry-over contamination If the negative-control PCR (without template DNA) shows a PCR product or a smear, exchange all reagents. Use disposable pipet tips containing hydrophobic filters to minimize cross-contamination. Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis
enzyme concentration too high When using HotStarTaq or Taq DNA Polymerase, use 2.5 units per 100 µl reaction.
too many PCR cycles Reduce the number of cycles in steps of 3 cycles.
Mg2+ concentration not optimal
Primer concentration not optimal or primers degraded
Leila Hosseinzadeh Anvar Thank you. I will try again with your advices.
Kazi Tawsif Ahmed Yuan-Yeu Yau Paul Rutland Thank you. I will try with your recommendations.
the concentration of the cDNA sample should be checked. And adding a positive and a negative control will also reveal the problem. try adjusting different annealing temperatures if above all are correct. you can do a gradient PCR for this.
Please check the total RNA concentration and adjust before running reverse transcription.
Please see the QIAGEN website (https://www.qiagen.com/us/resources/faq?id=4eb03cc8-4623-4e9e-96b2-6a4c17c03c58&lang=en), explaining all the possible factors and suggestions on how to avoid it.
I second Shirin Parizad suggestion of Qiagen. It is a common problem and the underlying reason is too much amplification is going on. Try to decrease the number of cycles.
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