I am trying to make a construct of murine Interferon beta (IFNb) fusing with a reporter gene. So, I wanted to amplify the IFNb gene. To do so I designed a primer and the primer had a flanking Restriction enzyme site. So, the primer was ~33mer in length and 18bp matched with the murine-IFNb sequence. Expected product size should be ~550bp. But when I am using this primer to do the PCR, I do not get the product band. I can say that the cDNA is okay, since beta-Actin control showed expected band. And I did a gradient PCR to check the various annealing temperatures, from a range of 51-59℃. I still didn't get any product band.
Please let me know if anyone has any suggestion.
Hi there,
I would go trying additives like DMSO (3% is the usual dose).
The other obvious reason for a negative result might be that your cDNA library is not covering the whole transcriptome.
And finally you are talking about a single primer which is weird as you actually need a pair of primers to get an efficient amplification.
A few things to try:
Double-check that your primers are designed to amplify only exons in your IFNb gene. If you designed primers based on genomic DNA, they may not work on cDNA.
Do you have cells that you KNOW express this gene? As Dominique said, your cell line might not express this gene.
Which end of the primer has your restriction site added? It needs to be at the 5' end of the primers or else it will block elongation.
Finally, use a broader range for your gradient PCR. Drop it down to 48 and as high as 65.
Good luck!
Thanks for all your inputs. I found out that the reason was due to less amount of mRNA to start with. Due to inadequate mRNA of interest, the cDNA was also less resulting in no amplification. Since, it is an inducible gene, I tried infecting the cells with virus before extracting the RNA. That led to proper amplification of the cDNA.
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