We cloned our gene of interest in pcDNA3.1(-) in between EcoRI and XhoI. In colony PCR (with primers used for amplify gene of interest), we got distinct amplicon of desired length. However, we didn't get desired amplicon from isolated plasmid (from that colony used for colony PCR). Even after double digestion with EcoR1 and XhoI, we didn't get any fragment of our desired amplicon.
What could be the possible reason?
Hi Rahim,
I run 1% agarose gel after colony PCR and got desired amplicon of 1100 bp. I also run colony PCR product of plasmid having no insert and in agarose gel got nothing.
Thank you Rahim.
Yes, I run negative control (colony PCR of Plasmid having no insert) but there is no band found.
Hello Sir,
As you have said you have run 1% agarose gel after colony PCR and got desired amplicon of 1100 bp. You also run colony PCR product of plasmid having no insert and in agarose gel got nothing. So it is better you can try to check the colony PCR result in RT PCR. Taq polymerase does not work on RNA samples, so PCR cannot be used to directly amplify RNA molecules. The incorporation of the enzyme reverse transcriptase (RT), however, can be combined with traditional PCR to allow for the amplification of RNA molecules. After you add your RNA sample to the PCR machine, add a DNA primer as usual and allow it to anneal to your target molecule. Then add RT along with dNTPs, which will elongate the DNA primer and make a cDNA copy of the RNA molecules and run the PRC reaction as usual. The product of RT-PCR is a double stranded DNA molecule analogous to the target segment of the RNA molecule.
- Badges
- Science method