Hello
I am trying to clone the G gene from Viral hemorrhagic septicemia virus.
For this purpose I cloned the G gene into pcDNA vector.
After that I transfected it into EPC cell line and check the success of transfection by fluorescence.
For confirming the transcription I did a RT-PCR and for confirming the translation I did western blot.
But I couldn't get any band from RT-PCR or from western blot.
Can someone help me to solve this problem?
Thanks.
More information is needed about your controls. I guess that you verified that the G gene was correctly cloned into the vector. What was fluorescing when you checked the success of transfection?
Since RT-PCR did not work, can you amplify by regular PCR using the same primers as with RT-PCR from the DNA from the transfected cells? Did you have a positive control for the RT-PCR, such as another gene on the plasmid, an antibiotic resistance gene, for example. Has your RT-PCR assay been previously tested? If not, there are many steps to troubleshoot an RT-PCR reaction (quality of RNA, does control gene amplify, try several primer sets, try different reverse transcription methods, one step vs two step reactions).
hello thanks for your reply
i used RFP and EGFP for confirmation of successful transfection.
for RT-PCR i used before after transfection cell with a dual vector and got the a band for the control gene (EGFP) and i got for the G gene but more weak band.
but for extraction of DNA from transfected cell i don't it will work because there will remaining of plasmid which contains the G gene.
thank you again for your help
Hello Adham,
By suggesting extracting DNA from the transfected cells, it was meant that you get the plasmid DNA because if you were getting no results this would be one way to verify that the G gene was in the cells and you could amplify it with the same primers.
But you have indicated that there is a faint band with your RT-PCR, so this suggests that you need to optimize your RT-PCR reaction.
Are RFP and EGFP on the same plasmid as the G gene? If so then they would have the same copy number as the G gene, also indicating that your RT-PCR may need to be optimized. If your control genes are not on the same plasmid then it would be good to test for something on the same plasmid as the best comparison for the G gene and copy number.
One thing to consider is whether your faint band of the G gene is from contaminating DNA in your RNA, and this is checked by using a no reverse transcriptase control in your reactions.
It seems that you are visualizing your results on a gel. If you can view them in real time and can get a curve of your amplification, the shape of the curve will give some information, such as does amplification begin late (indicates low copy number) or does it begin early but not amplify well (indicates poor reaction efficiency).
If you have a good signal in RT-PCR for EGFP that suggests your reverse transcription method is good. Then I would look at your specific primers and design a few sets of primers to optimize. You could also adjust PCR conditions, such as lower annealing temperature.
It is not an easy solution, but I hope this helps.
thank you so much for your suggestion. i will try to follow them and i will tell your the results.
- Badges
- Science method