I loaded PCR product on an agarose gel and I get a long smear. The starting material for PCR was cDNA. I repeated the experiment with fresh reagents but show same results. While the quantity of cDNA was more than 2000ng/ul. While I have also used low amount of template and primers but didn’t work. Does anyone have advice ?
- Are you sure the target is present in that cDNA?
- Check the quality of the starting RNA.
- Use a temperature gradient across the block to find out whether there is an optimal annealing temperature.
- Keep the number of cycles low -under 25.
When you say that using less starting template did not work, do you mean that there was again a smear or that there was no amplification? What did your no template control yield?
Hi Ayyaz,
working with cDNA means that your primers have been designed on the coding sequence of your gene. can you check on the UCSC in-silico PCR tool if it has been done or not (http://genome.ucsc.edu/cgi-bin/hgPcr).
fred
My Suggestion for resolve this problem put different Conc. MgCl2. this problem may resolved.
thank you Alejandro Martin the low concentration also shows smear while sir you may have observed in my gel picture that two samples show amplified product but stick in well do you have any solution for this. Sir this was a 25 cycle reaction you recommend me to decrease cycle number. sir next time will use temperature gradient and with different concentrations of MgCl2 as suggested by Kamlesh Kaitholia . Frederic Lepretre sir I am working on RNA virus and their genome is not present in given link by you.
Hi Ayyaz,
yes you can use temperature and MgCl2 gradients in the same run but first test your primers in NCBI website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) with the right genome.
fred
Can you provide more details in how the cDNA sample was obtained and how did you determine concentration? Also, how much did you add when using low amounts of template?
Alejandro Martin sir the cDNA was synthesized using this RevertAid First Strand cDNA Synthesis Kit (thermofisher) while for determining concentration I am using NanoDrop spectrometer. Firstly I was using 4 ul (con. more than 2000ng/ul)in 25 ul reaction now i am using 2 ul in 25 ul reaction but showing same results.
If you did not purify the cDNA then your nanodrop readings are thrown off by the free nucleotides/primers present in the RT reaction. The actual concentration is probably much lower.
Also, if you are going to try lower template concentrations go for 10- to 100-fold lower, not merely 2-fold.
Hope it helps!
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